Cerebrovascular miRNAs Track Early Development of Alzheimer’s Disease and Target Molecular Markers of Angiogenesis and Blood Flow Regulation

Abstract

Background:

Alzheimer’s disease (AD) is associated with impaired cerebral circulation which underscores diminished delivery of blood oxygen and nutrients to and throughout the brain. In the 3xTg-AD mouse model, we have recently found that > 10 cerebrovascular miRNAs pertaining to vascular permeability, angiogenesis, and inflammation (e.g., let-7d, miR-99a, miR-132, miR-133a, miR-151-5p, and miR-181a) track early development of AD. Further, endothelial-specific miRNAs (miR-126-3p, miR-23a/b, miR-27a) alter with onset of overall AD pathology relative to stability of smooth muscle/pericyte-specific miRNAs (miR-143, miR-145).

Objective:

We tested the hypothesis that cerebrovascular miRNAs indicating AD pathology share mRNA targets that regulate key endothelial cell functions such as angiogenesis, vascular permeability, and blood flow regulation.

Methods:

As detected by NanoString nCounter miRNA Expression panel for 3xTg-AD mice, 61 cerebrovascular miRNAs and respective mRNA targets were examined using Ingenuity Pathway Analysis for canonical Cardiovascular (Cardio) and Nervous System (Neuro) Signaling.

Results:

The number of targets regulated per miRNA were 21 ± 2 and 33 ± 3 for the Cardio and Neuro pathways respectively, whereby 14 ± 2 targets overlap among pathways. Endothelial miRNAs primarily target members of the PDE, PDGF, SMAD, and VEGF families. Individual candidates regulated by ≥ 4 miRNAs that best mark AD pathology presence in 3xTg-AD mice include CFL2, GRIN2B, PDGFB, SLC6A1, SMAD3, SYT3, and TNFRSF11B.

Conclusion:

miRNAs selective for regulation of endothelial function and respective downstream mRNA targets support a molecular basis for dysregulated cerebral blood flow regulation coupled with enhanced cell growth, proliferation, and inflammation.

INTRODUCTION

Alzheimer’s disease (AD) is the most prevalent form of dementia (~6.7 million people in the United States alone) [1] and is described by compromised neurovascular regulation of blood flow throughout the brain [2]. Clinical evidence now clearly demonstrates that cerebrovascular dysfunction encompassing intracranial arteries, arterioles, and capillaries results in decreased blood flow, reduced cerebral oxygenation, and increased white matter hyperintensities that altogether drive and accompany neurodegenerative AD pathology [3, 4]. Further, the prognosticative value among the intersection of cerebrovascular disease, neurodegeneration, and amyloidosis markers is widespread for determining cognitive decline among racially and ethnically diverse individuals [5, 6]. Despite such observations, only a small percentage (≤5%) of agents currently in clinical trials are dedicated to directly addressing vascular health versus ≈30% for amyloid and tau [7]. The molecular pathogenesis of AD remains obscure, whereby no effective prevention or treatment strategies have been secured yet. As a potential non-invasive diagnostic and therapeutic tool, vascular miRNAs that negatively regulate expression of downstream messenger RNA (mRNA) and protein targets [8] are promising biomarkers due to their novelty in modern molecular biology research while stable in the blood circulation [9]. We have recently found that cerebrovascular miRNAs track early development of AD pathology in the 3xTg-AD model [10].

If not stably expressed throughout, cerebrovascular miRNAs are primarily downregulated during AD relative to pre-AD conditions in the 3xTgAD model which include let-7d, let-7g, miR-23a, miR-23b, miR-27a, miR-29a, miR-29c, miR-99a, miR-126-3p, miR-132, miR-135a, miR-150, miR-151-5p, and miR-181a [10]. Upregulated miRNAs during AD include miR-29a, miR-29c, and miR-690. For sex-based differences, most of the downregulated miRNAs emerged in AD males (let-7d, let-7i, miR-23a, miR-34b-3p, miR-99a, miR-126-3p, miR-132, miR-150, miR-151-5p, miR-181a) with only two for females (miR-150, miR-539). This general finding aligns with the bulk of structural changes (density, junctions, and length) observed primarily in the cerebral vessels of male 3xTg-AD animals [11]. In addition, cerebrovascular miRNAs can delineate conditions of cognitive impairment versus young as let-7g and miR-1944 in males and miR-133a and miR-2140 in females [10]. With onset of extracellular amyloid- (A) deposition relative to cognitive impairment, miR-99a is downregulated in males. With the exception of miR-2141, all of the detected cerebrovascular miRNAs were stably expressed from the onset of AD and onward for progression of disease. Thus, a strength and limitation of cerebrovascular miRNA expression appears to be the sole ability to mark early (but not late) development of AD.

An additional noteworthy finding in 3xTg-AD mice is that endothelial-specific-miRNAs (miR23a/b [12], miR-27a [13, 14], and miR-126-3p [15, 16]) are downregulated in AD versus pre-AD conditions, whereas smooth muscle/pericyte-specific miRNAs (miR-143 [17] and miR-145 [12, 18]) remain stable throughout [10]. Upregulated downstream molecular targets of endothelium-specific miRNAs govern vascular integrity [13], angiogenesis [16, 19], and clearance of A [20]. Accordingly, such members of the phosphodiesterase (PDE) [21], platelet-derived growth factor (PDGF) [22], “mothers against decapentaplegic” (SMAD) [23], and vascular endothelial growth factor (VEGF) [24] gene families are integral to progression of AD pathology. At the same time, vascular miRNAs may interface with the brain parenchyma for processes of neuroinflammation as well [25]. Altogether, emerging evidence points to AD as a convergence of cerebrovascular and neurodegenerative disease [26].

In light of our recent molecular [10] and structural [11] findings for cerebral vessels in 3xTg-AD animals, the current study endeavored to ascertain mRNA targets downstream of miRNAs indicative of AD onset. A key objective was to determine whether there were mRNA targets shared among miRNAs that changed with AD pathology in 3xTg-AD animals versus others that were stably expressed throughout. In addition, we examined differences in such targets across biological sexes regardless of AD progression. With the premise that AD is a neurovascular disorder [2], our approach entailed a comprehensive Ingenuity Pathway Analysis (IPA) of 61 distinct cerebrovascular miRNAs along pathway strings for cardiovascular (Cardio) and nervous system (Neuro) signaling. More than 1,800 targets emerge for identification among the two pathways, whereby 455 are shared. The most prominent individual mRNAs for marking the presence of AD pathology while regulated by ≥ 4 cerebrovascular miRNAs include Cofilin 2 (CFL2), Glutamate Ionotropic Receptor N-Methyl-D-Aspartate Type Subunit 2B (GRIN2B), PDGFB, Solute Carrier Family 6 Member 1 (SLC6A1), SMAD3, Synaptotagmin 3 (SYT3), and Tumor Necrosis Factor (TNF) Receptor Superfamily Member 11b (TNFRSF11B). Further, CFL2, Histone Deacetylase 8 (HDAC8), Insulin Like Growth Factor 1 Receptor (IGF1R), ORAI Calcium Release-Activated Calcium Modulator 2 (ORAI2), PDGFD, Sirtuin 1 (SIRT1), and Vascular Endothelial Growth Factor A (VEGFA) distinguish among males and females during Pre-AD and/or AD, alongside members of the Heat Shock Protein 70 (HSPA1A/B), Sodium Voltage-Gated Channel (SCN2A/B and 8A) and SYT (SYT1/2/4) families. Altogether, miRNAs selective for regulation of endothelial function and respective downstream mRNA targets support a molecular basis for dysregulated cerebral blood flow regulation coupled with enhanced cell growth, proliferation, and inflammation. Accordingly, pathways associated with cerebrovascular miRNAs and their respective mRNA targets advance mechanistic and therapeutic insight for resolving early AD development.

METHODS

Animal care and use, cerebrovascular RNA isolation, and NanoString miRNA profiling

In accord with approval by the Institutional Animal Care and Use Committee of Loma Linda University and performed in accord with the National Research Council’s “Guide for the Care and Use of Laboratory Animals” (8th Edition, 2011), our original study [10] isolated and examined cerebrovascular miRNA from 24 3xTg-AD mice [27, 28]. Three males and three females were used throughout four groups as young (1–2 mo) [29], cognitive impairment (CI, 4–5 mo) [29], presence of extracellular Aβ plaques (Aβ, 6–8 mo) [28], and extracellular Aβ plaques with neurofibrillary tangles composed of tau (AβT, 12–15 mo) [28]. Note that these study groups are described by absence of cognitive deficits with no overt Aβ or tau throughout the entire brain (young) [29]; early deficits in memory retention coupled with intraneuronal A pathology in the hippocampus, cortex, and amygdala but no “plaques” as extracellular Aβ deposition (CI) [29]; moderate cognitive impairment coupled with cortical Aβ but no tau pathology (Aβ) [28]; and severe cognitive impairment coupled with extensive hippocampal inflammation (AβT) [28] respectively.

The purification of cerebral blood vessels from each mouse brain was based on a modified protocol originally developed by the Cohen-Salmon laboratory [30]. In brief, brain tissue was homogenized and then subjected to a series of centrifugation and filtration cycles in HEPES-buffered Hanks Balanced Salt Solution with bovine serum albumin. Following final RNA extractions from all animals/groups, samples were analyzed using a mouse miRNA nCounter platform (CSO-MMIR15-12, Mouse Version 1.5 miRNA assay; NanoString Technologies, Seattle, WA, USA). Using the nSolver software, (Version 4.0; NanoString Technologies) detected miRNAs with a normalized count threshold of ≥ 5 [31] across all samples were retained for further analysis. Details of respective protocols can be consulted in Chum et al. [10].

Signaling pathway and statistical analyses

Ingenuity Pathway Analysis (IPA; Qiagen, Germantown, MD, USA) microRNA Target Filter was used to generate miRNA:mRNA targeting information. 61 out of the 86 microRNAs uploaded have targeting information available in IPA. “Experimentally Observed” and “High (predicted)”were selected for miRNA Confidence filter. Ingenuity Canonical Pathways pertaining to Cardiovascular Signaling (Cardio) were selected and filtered to 61 microRNAs targeting 711 mRNAs. In addition, Neurotransmitter and Other Nervous System Signaling (Neuro) was selected and filtered to 61 microRNAs targeting 1163 mRNAs. Graph Pad Prism (Version 9; San Diego, CA, USA) was used for plotting data and conducting data analysis. For simultaneous comparison among pathology groups (young versus CI versus Aβ versus AβT or Pre-AD versus AD) and both sexes, statistical analysis included two-way Analysis of Variance (ANOVA) with Tukey’s post hoc correction for multiple comparisons. As applicable, Shapiro-Wilk and Kolmogorov-Smirnov parametric tests examined normal distribution of data combined among biological sexes as Young versus CI versus Aβ versus AβT (n=6 each). For overall Pre-AD (Young + CI) and AD (Aβ versus AβT) datasets where n=12 per group, Anderson-Darling and D’Agostino and Pearson parametric tests were additionally applied. Kruskal-Wallis (Dunn’s correction for multiple comparisons) and Mann-Whitney tests were used in replacement of one-way ANOVA (Holm-Sidak posthoc) and two-tailed unpaired t-tests respectively for non-parametric comparisons. Note that a combination of young with CI for Pre-AD grouping was for the purpose of gauging the indicative sensitivity of various miRNAs (individual group transitions and overall AD versus Pre-AD) and how advanced pathology needs to be in order for certain changes in miRNA expression to take place. Outlying data points were determined by the robust regression and outlier removal (ROUT, Q=1%) method in comparisons that involved n≥6 per study group. An n of 1 represented one independent miRNA expression analysis from one mouse brain. All summary data are presented as the mean±SEM and differences among groups were accepted as statistically significant with p<0.05.

RESULTS

The primary goal of the current study was to determine individual mRNA targets downstream of miRNAs that are altered versus those that were stable in expression upon onset of AD in 3xTg-AD mice [10]. Further, we also examined whether various targets aligned with each biological sex (males and females) during Pre-AD and/or AD conditions. A total of 61 separate miRNAs with distinct pools of targets were recognized and mapped by the IPA canonical Cardio and Neuro pathway strings. Presentation of data is organized as an overview of the number of mRNAs targets per miRNA that are recognized by Cardio, Neuro, and overlap of Cardio and Neuro (Figs. 1 and 2). Representative Cardio and Neuro Venn diagrams of mRNA targets are illustrated for miRNAs that best mark AD pathology for Figs.3–7.Table 1 provides an overview of all 61 miRNAs as points of significance (or the lack thereof) among AD pathology and/or biological sex alongside lists of all individual mRNA targets for both Cardio and Neuro. Supplementary Tables 1 and 2 list all individual mRNA targets for Cardio and Neuro respectively with lists of all regulating miRNAs per target. Table 2 illustrates distinguished mRNA targets shared by≥3 miRNAs with scoring for indicative strength of AD pathology and biological sex. Finally, Tables 3 and 4 present a final distillation of the most prominent mRNA markers (+ known cellular roles) for AD pathology and biological sex respectively.

Fig. 1.

Total number of mRNA targets among the Cardiovascular Signaling (Cardio), and Neurotransmitter and Other Nervous System Signaling (Neuro) pathways per each of the cerebrovascular miRNAs expressed in 3xTg-AD mice. The Cardio and Neuro pathway strings revealed a total of 711 and 1,163 mRNA targets respectively, whereby each miRNA has 21±2 (Cardio) and 33±3 (Neuro) targets. The overlap among respective pathways strings revealed a total of 455 mRNA targets, whereby each miRNA shares 14±2 targets. See Supplementary Tables 1 and 2 for a complete list of Cardio and Neuro targets respectively along with their regulating miRNAs.

Fig. 2.

Fraction of mRNA targets overlapping among the Cardiovascular Signaling (Cardio) and Neurotransmitter and Other Nervous System (Neuro) pathway strings. A) Fraction of overlap among Cardio (red) and Neuro (blue) pathways respectively per each cerebrovascular miRNA expressed in 3xTg-AD mice. Overall, the fraction of Cardio targets (0.66±0.02) shared with Neuro is significantly higher (p<0.05,unpaired t) than the fraction of Neuro targets (0.41±0.01) shared with Cardio. B) Fraction of overlap among Cardio (red) and Neuro (blue) pathways for cerebrovascular miRNAs that altered with AD pathology versus miRNAs that were stable throughout. Note that the fraction of shared Cardio targets is significantly higher (p<0.0001, two-way ANOVA) than that of Neuro throughout for cerebrovascular miRNAs that changed in expression with AD (Cardio, 0.68±0.03>Neuro, 0.41±0.02) versus miRNAs that were stable throughout (Cardio, 0.66±0.02>Neuro, 0.40±0.02). *p<0.05, Cardio>Neuro, two-way ANOVA.

Fig. 3.

Overlap of mRNA targets among mmu-miR-let-7d, mmumiR-181a, mmu-miR-132, and mmu-miR-99 as top indicators of the AD pathology. Bold text: precise targets that overlap among respective miRNAs; italicized text: similar isoforms among the same gene family. A) Cardio: Note precise overlap for KRAS, IGF1R, TNFSF10, and TNFRSF11B. Shared families among at least two miRNAs include DNAJ, FGF, FZD, IGF, IL, MMP, PRK, RAS, RHO, and TNFSF. B) Neuro: Note precise overlap for GRIN2B, KRAS, IGF1R, and POLR2D. Shared families among at least two miRNAs include ADAMTS, BCL2, FZD, GPR, IGF, IL, MMP, POLR2, PRK, RAS, RHO, ROR, SLC, and TGFB. See Table 1 for all details regarding each miRNA and original data are reported in [10].

Table 1.

Significance of Alzheimer’s disease (AD) pathology among 61 miRNAs and respective mRNA targets as indicated via the Cardiovascular Signaling (Cardio) and Neurotransmitter and Other Nervous System Signaling (Neuro) pathway strings. The Cardio and Neuro pathway strings revealed 711 and 1163 mRNA targets respectively (overlap = 455 targets)

miRNA	AD pathology Significance	Biological Sex Significance	mRNA Targets Cardiovascular System	mRNA Targets Nervous System	Overlapped mRNA Targets
mmu-let-7d	Aβ versus Young (Male) Aβ and AβT versus Young AD versus Pre-AD (Male) AD versus Pre-AD	—	ACVR1C, ADRB2, ADRB3 AKAP8, AQP4, ARG2, ARHGEF38, CACNG4, CALM1, CASP3, CCND1, CDC34, CDKN1A, CHP1, CHRNA7, COL1A1, COL1A2, CSNK1D, DIAPH2, DNAJC1, DVL3, EDN1, ELP1, F2, F2RL3, FASLG, FGF4, FZD3, GNG5, HAND1, HIF1AN, HMOX1, ICMT, IGF1R, IL12A, IL13, ITGB3, KLK10, KRAS, MAP3K13, MAPK6, MYC, NEDD4, NRAS, PDE12, PDGFA, PDGFB, PLA2G2F, PLA2G3, PLD3, PPP1R7, PRKAR2A, PTGS2, RAS, RHOB, RHOG, RPS6KB2, S100A8, TGFBR1, TGFBR2, TGFBR3, THBS1, TNFSF10, TNFSF9, TP53, UTRN, VIM, WNT1, WNT9A	ACP1, ACVR1C, ADAMTS12, ADAMTS14, ADAMTS15, ADAMTS2, ADAMTS8, ADGRG1, ADRB2, ADRB3, ARHGAP8, ARHGAP8-PRR5, ARHGEF38, BCL2L1, CACNG4, CALM1, CASP3, CCL3, CCND1, CCR7, CD200R1, CHP1, CSNK1D, DRD3, ELP1, F2RL3, FAS, FASLG, FZD3, GNG5, GPR157, GPR26, GPR63, GRIN2B, HMOX1, HTR1E, IFNA4, IGF1R, IL10, IL12A, ITGB3, KCNJ11, KCNJ16, KRAS, LAMC1, LIMK2, LIPH, MAP3K13, MAPK6, MYC, NGF, NRAS, PAPPA, PDGFA, PDGFB, PLA2G2F, PLA2G3, POLR2C, POLR2D, PPP1R7, PRKAR2A, PTAFR, PTGS2, RAS, RGS16, RHOB, RHOG, RPS6KB2, SCN11A, SCN4B, SCN8A, SIGMAR1, SLC1A4, SLC38A1, SMOX, SNAP23, TAF9B, TGFBR1, TGFBR2, TGFBR3, THBS1, TLR4, TP53, TUBB4A, UTRN, VCAN, WNT1, WNT9A, XCR1	ACVR1C, ADRB2, ADRB3, ARHGEF38, CACNG4, CALM1, CASP3, CCND1, CHP1, CSNK1D, ELP1, F2RL3, FASLG, FZD3, GNG5, HMOX1, IGF1R, IL12A, ITGB3, KRAS, MAP3K13, MAPK6, MYC, NRAS, PDGFA, PDGFB, PLA2G2F, PLA2G3, PPP1R7, PRKAR2A, PTGS2, RAS, RHOB, RHOG, RPS6KB2, TGFBR1, TGFBR2, TGFBR3, THBS1, TP53, UTRN, WNT1, WNT9A
mmu-miR-181a	Aβ and AβT versus Young (Male) CI, Aβ, and AβT versus Young AD versus Pre-AD (Male) AD versus Pre-AD	—	BCL2, ESR1, GATA6, HSP90B1, IL1A, IL2, IL25, KRAS, LTB, MMP14, PRKCD, RHOH, SLC9A3, TNFRSF11B, TNFSF10	ADAMTS5, AFG3L2, ARF6, BCL2, BHLHE40, CDKN1B, ENTPD6, GABRA1, GRIA2, GRIN2B, HLA-A, HSP90B1, IL1A, ITSN1, KRAS, LRRC8D, MMP14, PRKCD, PRKN, RHOH, RORB, S1PR1, SCD, TCERG1	BCL2, HSP90B1, IL1A, KRAS, MMP14, PRKCD, RHOH
mmu-miR-132	Aβ and AβT versus Young (Male) Aβ versus Young AD versus Pre-AD (Male) AD versus Pre-AD	Pre-AD	EP300, IL11, IL36B, MMP9, PTEN, PTPA, RASA1, SMAD9, TGFB2, TNFRSF11B, TNFSF4, UBE2D2	BTC, CAPN8, CFL2, EP300, GPR89A, GPR89B, GRM3, IL36B, LIPM, MMP9, NFE2L2, POLR2D, POLR2J, PSMA2, PSMD12, PTEN, PTPA, RASA1, SIRT1, SLC6A1, SMAD9, TGFB2	EP300, IL36B, MMP9, PTEN, PTPA, RASA1, SMAD9, TGFB2
mmu-miR-99	miR-99a Aβ and AβT versus CI (Male) Aβ versus CI AD versus Pre-AD (Male) AD versus Pre-AD	miR-99b Young Pre-AD	DNAJB7, FGF16, FGF21, FGFR3, FZD8, IGF1R, MTOR, PPP3CA, SELE, SMPDL3B	ADGRE2, EIF4EBP2, FGFR3, FZD8, GPR141, IGF1R, LRRC8B, MTOR, NLRP2, NTRK3, PPP3CA, PTGIR, RORA, RPTOR, SCN1B, SMARCA4	FGFR3, FZD8, IGF1R, MTOR, PPP3CA
mmu-miR-151-5p	Aβ versus Young AD versus Pre-AD (Male) AD versus Pre-AD	—	AKT2, CACNG7, CXCL12, DNAJC22, IL36RN, ITGA10, MAP3K20, MAPK11, PPP1R12B, RALB, TNNT2	AKT2, APH1A, CACNG7, CHMP1A, CHRM2, CXCL12, DNM1, DOK1, DOK7, EFNB2, FKBP1A, FOSB, GABRB1, GDNF, GPR173, GRIN1, IL36RN, ITGA10, KCNJ10, LHCGR, MAPK11, MPZ, P2RX6, PPP1R12B, PTGDR2, RAB35, RALB, RXFP3, S1PR3, SYT3, TRPV4, VAMP2	AKT2, CACNG7, CXCL12, IL36RN, ITGA10, MAPK11, PPP1R12B, RALB
mmu-miR-23	miR-23a AD versus Pre-AD (Male) AD versus Pre-AD miR-23b AD versus Pre-AD	miR-23a, AD miR-23b, Pre-AD	ACVR1C, CXCL12, HIF1AN, HK1, IL18, IL6R, LDHB, LYZ, MET, MYH1, MYH4, MYL12B, PDE4B, PDE7A, PKIA, PTEN, RRAS2, SMAD3, SMAD4, SMAD5	2700046G09Rik, ACVR1C, APAF1, CCK, CDH17, CFL2, CXCL12, FAS, GABRA6, IL18, IL6R, LMNB1, MARCKS, MET, MYH1, MYH4, MYL12B, NOTCH1, NRP1, PDE4B, PTEN, PTGDR, RGS18, RGS8, RRAS2, SMAD3, SMAD4, SMAD5, SNAP25, SST, STXBP6, SYT4, VTI1B	ACVR1C, CXCL12, IL18, IL6R, MET, MYH1, MYH4, MYL12B, PDE4B, PTEN, RRAS2, SMAD3, SMAD4, SMAD5
mmu-miR-126-3p	AD versus Pre-AD (Male) AD versus Pre-AD	—	IL22RA2, IRS1, ITGA11, LPAR2, MEF2B, PIK3R2, PRKG1, VCAM1, VEGFA	ADAM9, CRKL, IRS1, ITGA11, KCNJ1, LPAR2, MEF2B, PIK3R2, PLXNB2, PRKG1, RGS3, SOX2, SYT8, TSC1, VCAM1, VEGFA	IRS1, ITGA11, LPAR2, MEF2B, PIK3R2, PRKG1, VCAM1, VEGFA
mmu-miR-150	AD versus Pre-AD (Male and Female) AD versus Pre-AD	Pre-AD and AD	AKT, CEBPB, DNAJB7, HDAC8, HSPA5, MYH1, NPR3, PDGFB, PDIA3, PTK2, RCAN1, SP1, VEGFA	AKT, ARRB1, CSF1R, EGR2, EREG, GRIN2B, HDAC8, HSPA5, MYH1, NPR3, PDGFB, PDIA3, PSMC1, PTK2, SCN2B, SP1, VEGFA	AKT, HDAC8, HSPA5, MYH1, NPR3, PDGFB, PDIA3, PTK2, SP1, VEGFA
mmu-miR-27a	AD versus Pre-AD	—	ACTA2, AKAP7, AQP11, BAX, CACNA2D3, CACNG2, CSF1, DNAJC13, FGF1, FOXO1, GAB1, GNG13, GRB2, GSK3B, IFNG, IGF1, LPAR6, LRRC55, MAP2K4, MAPKAPK2, MEF2C, MMP13, MYL12A, NEDD4, PDE6D, PDE7B, PDPK1, PKIA, PLCL2, PPARG, PPP1CC, RCAN2, RPS6KA5, RXRA, SHE, SMAD3, SMAD4, SMAD5, SMAD9, UBE2D1, UBE2N, UBE2V1, VEGFC	ACTA2, ADORA2B, BAX, CACNA2D3, CACNG2, FASN, FGF1, FOSB, FOXO1, FSHB, GAB1, GNG13, GOSR2, GPR174, GRB2, GRIA4, GRIN2D, GRK4, GSK3B, HBEGF, HCRTR1, HTT, IFNG, IGF1, ITSN2, LIMK1, LIPH, LPAR6, LRRC55, MAP2K4, MEF2C, MMP13, MYL12A, NMUR2, NOTCH1, NOTCH2, NRP2, NRXN3, PDE6D, PDK4, PDPK1, PLCL2, PPP1CC, PSMA1, PXN, RGS1, RGS8, RPS6KA5, SEMA7A, SHE, SLC6A1, SMAD3, SMAD4, SMAD5, SMAD9, SNAP25, SPRY2, STMN2, SYT3, TPH2, VEGFC, VIP	ACTA2, BAX, CACNA2D3, CACNG2, FGF1, FOXO1, GAB1, GNG13, GRB2, GSK3B, IFNG, IGF1, LPAR6, LRRC55, MAP2K4, MEF2C, MMP13, MYL12A, PDE6D, PDPK1, PLCL2, PPP1CC, RPS6KA5, SHE, SMAD3, SMAD4, SMAD5, SMAD9, VEGFC
mmu-miR-135a	AD versus Pre-AD	—	ANGPT2, APC, BMPR1A, CACNA1E, CAMK1G, CASP1, CCNG2, CD40LG, CLCF1, DNAJC9, EDNRA, FGF11, FOXO1, JAK2, KCNN3, MEF2A, MEF2C, NR3C2, PDE1A, PDE8B, PIK3R2, RAP2A, SMAD5, SNTB2, TCF7L2, TNFRSF11B, WNT3	ABAT, ADCYAP1, APC, BMPR1A, CACNA1E, CAMK1G, CASP1, CPLX1, CPLX2, CSNK1G2, CXCL10, EDNRA, ENTPD4, ENTPD7, FKBP1A, FOXO1, GPR21, GPR55, GRIN2B, HTR1A, JAK2, KCNJ6, KCNN3, MEF2A, MEF2C, NEFM, NTNG1, OPN5, PDE1A, PIK3R2, PTGER1, RAP2A, SLC6A4, SMAD5, SYT2, SYT3, TCF7L2, TRPC6, WNT3	APC, BMPR1A, CACNA1E, CAMK1G, CASP1, EDNRA, FOXO1, JAK2, KCNN3, MEF2A, MEF2C, PDE1A, PIK3R2, RAP2A, SMAD5, TCF7L2, WNT3
mmu-miR-133a	CI and AβT versus Young (Female) AβT versus Young	Young	CCN2, CDC42, CRK, DUSP1, FGF1, FGF12, FGFR1, IGF1R, KCNH2, KCNMB1, MAP3K2, NFATC4, PDE1C, PDE8B, PKM, PPP2CA, PPP2CB, PPP2R2D, RHOA, SGK1, SRF, SUMO1, TFAP2D, UBE2Q1, UBE2Q2	ARFIP2, ARRB1, CD200, CDC42, CLTA, CRK, EFNA4, FGF1, FGFR1, GALR1, GCH1, GDNF, GNGT2, GPR84, HLA-DQA1, IGF1R, KCNH2, KCNMB1, MAP3K2, MC2R, NDRG1, NFATC4, NKX2-2, P2RX4, PAPPA, PDE1C, PER2, PFN2, PPP2CA, PPP2CB, PPP2R2D, PTH1R, RGS21, RHOA, RIMS1, SCN2B, SGK1, SLC52A2, SLC6A1, SRF, SYT1, SYT2, TUBB1, VAMP2, YES1	CDC42, CRK, FGF1, FGFR1, IGF1R, KCNH2, KCNMB1, MAP3K2, NFATC4, PDE1C, PPP2CA, PPP2CB, PPP2R2D, RHOA, SGK1, SRF
mmu-miR-690	Aβ versus Young and CI AD versus Pre-AD	—	CASP14, CTNNB1, KLK7, RELA, RRAS2	CASP14, CRP, CTNNB1, GPR22, ITSN1, RELA, RRAS2	CASP14, CTNNB1, RELA, RRAS2
mmu-miR-29c	AD versus Pre-AD	—	ACVR2A, CASP8, CDC42, COL1A1, COL1A2, COL2A1, COL3A1, COL5A3, EDNRB, GNG12, GPR37, HDAC4, IL36G, LEP, P2RY4, PDGFA, PDGFB, PDGFC, PIK3R1, PPP1R3D, PTEN, REL, RND3, SGK1, SP1, TFAP2C, TGFB3, TGFBR1, TGFBR2, TNFRSF1A, TPM1, VEGFA	ACVR2A, ADAM19, ADAMTS10, ADAMTS12, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS2, ADAMTS20, ADAMTS7, ADAMTS9, ARPC3, ATP5MC1, BACE1, BET1L, CASP8, CDC42, CNR1, CYCS, DCX, DPYSL5, EDNRB, EFNA5, ENTPD7, ETV4, GABRP, GLIS2, GNG12, GPR156, GPR37, GPR82, GRIP1, HDAC4, IL36G, LAMA2, LAMC1, NAPB, P2RY4, PDGFA, PDGFB, PDGFC, PER3, PIK3R1, PLP1, PPP1R3D, PTEN, REL, RGS1, RND3, RNF19A, ROBO1, SGK1, SP1, STMN2, TGFB3, TGFBR1, TGFBR2, TNFRSF1A, TRAF4, TUBB2A, VCAN, VEGFA, YY1	ACVR2A, CASP8, CDC42, EDNRB, GNG12, GPR37, HDAC4, IL36G, P2RY4, PDGFA, PDGFB, PDGFC, PIK3R1, PPP1R3D, PTEN, REL, RND3, SGK1, SP1, TGFB3, TGFBR1, TGFBR2, TNFRSF1A, VEGFA
mmu-miR-34b-3p	AD versus Pre-AD (Male)	Pre-AD	DNAJA1, GNG5, KCNMB3, P2RY1	CFL2, GABRA4, GNG5, GPR82, KCNMB3, P2RY1, PTGER4, RGS20	GNG5, KCNMB3, P2RY1
mmu-miR-129-3p	AβT versus CI	—	AKAP3, BAG3, BDKRB2, CACNA1D, CASP6, DVL3, EGLN3, HSPH1, IL12B, IL17A, MAPK3, MMP16, PKIA, PLA2G2F, PNPLA3, PRKCE, RCAN2, RHOU, SMAD3	ADAM8, BACE1, BDKRB2, CACNA1D, CASP6, EFNA1, GPR160, GPR39, GPR55, GRIN2B, IL12B, KCNJ1, MAPK3, MMP16, PLA2G2F, PNPLA3, PRKCE, RGS16, RGS17, RHOU, SCN3B, SMAD3, TICAM2	BDKRB2, CACNA1D, CASP6, IL12B, MAPK3, MMP16, PLA2G2F, PNPLA3, PRKCE, RHOU, SMAD3
mmu-miR-539	AD versus Pre-AD (Female)	Pre-AD	APEX1, GUCY1A2, ITPR1	GABRA4, GUCY1A2, ITPR1, KCNJ6, PTH, TBK1	GUCY1A2, ITPR1
mmu-miR-145	—	Aβ Pre-AD and AD	ACTB, ACTG1, AKAP9, CAMK1D, CCNA2, CMA1, EIF4E, ELP1, FGF5, IGF1R, IRS1, ITGB8, MAP3K1, MAPK7, MDM2, MMP1, MYC, PDGFD, PLCE1, PPP3CA, RASA1	ACTB, ACTG1, BIRC2, CAMK1D, CYCS, DDC, EIF4E, EIF4EBP2, ELP1, GLIS1, HOMER1, IFNGR2, IGF1R, IRS1, ITGB8, MAP3K1, MAPK7, MDM2, MMP1, MYC, NTN4, PDGFD, PLCE1, PPP3CA, PTGR2, RAB27A, RASA1, RGS7, SEMA3A, SRGAP2	ACTB, ACTG1, CAMK1D, EIF4E, ELP1, IGF1R, IRS1, ITGB8, MAP3K1, MAPK7, MDM2, MMP1, MYC, PDGFD, PLCE1, PPP3CA, RASA1
mmu-miR-146a	—	AβT AD	ATF6, CASP7, CCL5, CCNA2, CCR3, CD40, CFH, CHUK, CXCL8, CXCR4, DNAJC12, FZD3, HDAC8, HSPA1A, HSPA1B, IL12RB2, IL17A, IL1F10, IL1R1, IL1RL2, IL36A, IL36B, IL36G, IL36RN, IL37, KRAS, LTB, MMP16, NOS2, STAT1, UBE2G1	ARF6, BIRC7, BRCA1, CASP7, CCK, CCL5, CCR3, CD40, CHUK, CPLX4, CRP, CXCL8, CXCR4, FZD3, HDAC8, HLA-A, HSPA1A, HSPA1B, IFNA1, IFNA13, IFNB1, IL10, IL1F10, IL1R1, IL36A, IL36B, IL36G, IL36RN, IL37, IRAK1, IRAK2, KRAS, MMP16, MR1, NLGN1, NOS2, PDGFRA, POLR2D, PSMA4, PTAFR, RAB11A, SLC1A1, STAT1, SYT1, SYT14, TLR1, TLR10, TLR4, TLR9, TRAF6	CASP7, CCL5, CCR3, CD40, CHUK, CXCL8, CXCR4, FZD3, HDAC8, HSPA1A, HSPA1B, IL1F10, IL1R1, IL36A, IL36B, IL36G, IL36RN, IL37, KRAS, MMP16, NOS2, STAT1
mmu-miR-377	—	Young Pre-AD	ARHGEF37, ARHGEF38, ATM, CCNG2, CDC34, DNAJB9, DNAJC19, GNG10, GNG5, GSK3B, HDAC8, HMOX1, HSPB8, IL17RA, IL18R1, IL3, ITGA6, KRAS, LPL, LTB, LYZ, MAP3K8, MAPK10, MMP13, MYL6, PDE1A, PDPK1, PKIA, PNPLA2, PPP1R3C, PRKAB2, PRKG1, RAP2A, RCAN2, RHOB, RXRG, SHC3, UBE2D2, UBE2H, UBE2L3, UBE2V2, WNT9A	ADAMTS9, AREG, ARHGEF37, ARHGEF38, ARRB1, BCL2L1, BIRC7, CCR8, CDH6, CDKN1B, FBXL21P, GAL, GNG10, GNG5, GOSR2, GPR101, GRIN2B, GSK3B, H3-3A, H3-3B, HDAC8, HMOX1, HTR1B, ITGA6, KRAS, LAMB2, LPL, LRP8, MAP3K8, MAPK10, MMP13, MYL6, NOTCH2, NPBWR1, PDE1A, PDK3, PDK4, PDPK1, PNPLA2, PPP1R3C, PRKAB2, PRKG1, RAP2A, RAPGEF3, RGS2, RGS4, RHOB, RORA, RXRG, SCN4A, SCN8A, SHC3, STX16, STX1B, SYT1, SYT2, UNC5A, WNT9A, XIAP	ARHGEF37, ARHGEF38, GNG10, GNG5, GSK3B, HDAC8, HMOX1, ITGA6, KRAS, LPL, MAP3K8, MAPK10, MMP13, MYL6, PDE1A, PDPK1, PNPLA2, PPP1R3C, PRKAB2, PRKG1, RAP2A, RHOB, RXRG, SHC3, WNT9A
mmu-miR-101b	—	Young	ARG2, GNG12, MEF2A, P2RY12, UBE2D1, UBE2D2, UBE2V1	APP, CXCL10, GNG12, GPR183, ID4, MEF2A, NFE2L2, P2RY12, PTH, RGS17	GNG12, MEF2A, P2RY12
mmu-miR-204	—	Young	BMP1, CACNA2D4, CACNG2, CAMK1, CHP1, CREB3L4, CXCR2, DNAJC13, DNAJC14, DNM2, DVL3, FGF20, IL11, IL1RL1, ITGB4, MEF2C, MMP3, MMP9, NOSTRIN, PDE3A, PIK3CB, PRKAB2, SHC1, TGFBR2	ADGRE3, AP2A2, ARPC1B, BIRC2, BMP1, CACNA2D4, CACNG2, CAMK1, CD200, CDH11, CHP1, CREB3L4, DNM2, EFNB1, EFNB3, EGR1, EPHA5, EPHA7, EPHB6, GPR52, GPR6, GRIN2B, HLA-DRB5, ITGB4, MEF2C, MMP3, MMP9, NAPG, NLRP2, NR3C1, PDE3A, PIK3CB, POLR2K, PRKAB2, PSME1, RAB22A, SCN2A, SHC1, TGFBR2, VN1R1	BMP1, CACNA2D4, CACNG2, CAMK1, CHP1, CREB3L4, DNM2, ITGB4, MEF2C, MMP3, MMP9, PDE3A, PIK3CB, PRKAB2, SHC1, TGFBR2
mmu-miR-16	—	Pre-AD	ACVR2A, ACVR2B, ADSS2, AKT3, ALOX12, APLN, AQP11, ATF6, BCL2, CACNA2D1, CASP5, CCND1, CD40, CDC42, DNAJB4, EDA, EGFR, EGLN2, EIF2B2, EIF4E, F2, FGF18, FGF2, FGF7, FGFR1, GNAI3, GRB2, HGF, HMOX1, HSP90B1, HSPA1A, HSPA1B, HSPA4L, ICMT, IFNG, IGF1, IGF1R, IKBKB, IL10RA, IL15, IL31RA, IL7R, ITGA2, JUN, KCNMB1, KCNN4, MAP2K1, MAP2K4, MAPK3, MAPK8, MKNK1, MYLK, NPR3, PLD1, PLD3, PPP2R5C, PRKAR2A, PTGS2, RAF1, RHOT1, SGK1, SLC12A2, SLC2A14, SLC2A3, SLC7A1, SLC9A6, SLC9A8, SNTB2, TFAP2D, TNFSF13B, TNFSF9, UBE2G2, UBE2Q1, UBE2S, UBE2V1, VEGFA, WNT2B, WNT3A, WNT7A	ACTR2, ACVR2A, ACVR2B, AKT3, ARHGDIA, BCL2, BDNF, BTRC, CACNA2D1, CADM1, CAPNS1, CASP5, CCND1, CD200R1, CD40, CD80, CDC42, CDK5R1, CFL2, CLOCK, CRKL, EGFR, EIF4E, ENTPD7, FASN, FGF2, FGFR1, FSHB, GABBR1, GNAI3, GPR171, GPR62, GPR63, GRB2, H3-3A, H3-3B, HGF, HLA-DQB2, HMOX1, HSP90B1, HSPA1A, HSPA1B, HTR2A, HTR4, IFNG, IGF1, IGF1R, IGF2R, IKBKB, INSR, ITGA2, JUN, KCNJ2, KCNMB1, KCNN4, KLC1, LAMC1, LDAH, MAP2K1, MAP2K4, MAPK3, MAPK8, MKNK1, MYLK, NAPG, NCS1, NECTIN1, NOTCH2, NPR3, PAFAH1B1, PAFAH1B2, PANX1, PAPPA, PDK4, PNOC, POLR2H, PPP2R5C, PRKAR2A, PSMD7, PTGS2, PTH, RAB9B, RAF1, RGS5, RGS8, RHOT1, RPS6KA3, RTN4, SCN2A, SCN3A, SCN8A, SEMA6D, SGK1, SLC38A1, SLIT2, SMAD7, SMPD1, SNCG, SYT3, SYT4, TRAF3, UBE2S, VEGFA, VTI1B, WIPF1, WNT2B, WNT3A, WNT7A, XCR1	ACVR2A, ACVR2B, AKT3, BCL2, CACNA2D1, CASP5, CCND1, CD40, CDC42, EGFR, EIF4E, FGF2, FGFR1, GNAI3, GRB2, HGF, HMOX1, HSP90B1, HSPA1A, HSPA1B, IFNG, IGF1, IGF1R, IKBKB, ITGA2, JUN, KCNMB1, KCNN4, MAP2K1, MAP2K4, MAPK3, MAPK8, MKNK1, MYLK, NPR3, PPP2R5C, PRKAR2A, PTGS2, RAF1, RHOT1, SGK1, UBE2S, VEGFA, WNT2B, WNT3A, WNT7A
mmu-miR-22	—	Pre-AD	AKT3, ALOX15B, BMP7, DNAJB5, DNAJC27, ESR1, F5, GNAI3, IL13RA1, IL17RD, IL31, KCNMB1, KLK12, MAPK14, MAX, NCOA1, ODF1, PDE1A, PIK3CD, PRKAR2A, PRKCB, PTEN, RHOV, SLC2A1, SLC9A5, SRF, VCAM1, WNT2B	ADGRE5, ADORA1, AKT3, ARFIP2, ARPC5, ARRB1, BMP7, CBL, CD80, CDH15, CSF1R, CSPG4, EIF4EBP3, ERBB3, GHRHR, GLIS2, GNAI3, GPR132, GPR31, GPR55, GRM5, H3-3A, H3-3B, HOMER1, HRH1, KCNMB1, LAMC1, LTB4R, MAPK14, MC1R, MFGE8, NDEL1, NLRP3, NTRK2, ORAI2, PDC, PDE1A, PIK3CD, PRKAR2A, PRKCB, PSEN1, PTEN, RAB5B, RAPGEF3, RCOR1, RGS10, RGS2, RHOV, SIRT1, SLC2A1, SLC6A13, SRF, TACR3, TLR8, TUBB1, VASP, VCAM1, WNT2B	AKT3, BMP7, GNAI3, KCNMB1, MAPK14, PDE1A, PIK3CD, PRKAR2A, PRKCB, PTEN, RHOV, SLC2A1, SRF, VCAM1, WNT2B
mmu-miR-34c	—	Pre-AD	ADRA1D, AGTR1, ALOX12B, ASIC2, BCL2, BMP3, CACNB3, CACNG8, CASP2, CCND1, CD47, CREB1, CREB3L2, CTNNB1, DNAJC16, DNAJC24, F2RL2, F8, FGF23, FGF7, FICD, GNAO1, GNAQ, GNAS, HDAC1, HSPA1A, HSPA1B, HSPB6, IKBKE, IL6R, KCNJ8, LDHA, LEF1, LPAR2, LRP6, LRRC55, MAP2K1, MAP3K3, MAPK13, MET, MRAS, MYC, MYH9, NOTUM, P2RY2, PIP5K1A, PKIA, PLA2G2F, PLN, PPARG, PPP1R14D, PPP2R1A, PPP2R3A, PPP2R5A, PRKAB1, PRKCQ, PTPA, RASD2, RCAN1, RCAN3, RHOJ, ROCK1, RRAS, SCNN1G, SLC9A3, SMAD3, SNTB2, TGFB3, TGFBR1, THBS1, TNFSF14, TP53, UBE2L3, VEGFA, WNT1, WNT3	ACKR1, ADAM10, ADRA1D, AGTR1, AP2A2, AREG, ARHGAP1, ASIC2, BCAN, BCL2, BMP3, CACNB3, CACNG8, CALB1, CALCR, CAPN6, CASP2, CCKAR, CCND1, CCR1, CHD8, CPLX2, CREB1, CREB3L2, CSF1R, CTNNB1, CXCL10, DNM1L, DPYSL4, EFNB1, EPHA4, F2RL2, FARP2, GABRA3, GABRB1, GCH1, GFRA3, GNAO1, GNAQ, GNAS, GPR101, GPR22, GPRC5A, GRIN2B, HDAC1, HSPA1A, HSPA1B, HTR2C, IKBKE, IL6R, KCNJ8, LEF1, LGR4, LPAR2, LRRC55, MAP2K1, MAP3K3, MAPK13, MAPT, MET, MRAS, MYC, MYH9, NDRG1, NECTIN1, NMUR1, NOS1AP, NOTCH1, NOTCH2, NOTUM, NTN1, NTRK3, OXT, P2RX6, P2RY14, P2RY2, PDGFRA, PENK, PER2, PIP5K1A, PLA2G2F, PNOC, POLR2I, POLR2J2/POLR2J3, PPP1R14D, PPP2R1A, PPP2R3A, PPP2R5A, PRKAB1, PRKCQ, PTPA, RASD2, RCVRN, RGS17, RGS8, RGS9BP, RHOJ, ROCK1, RPS6KA4, RRAS, S1PR3, SCN2B, SCNN1G, SEMA4B, SEMA4D, SIRT1, SLC6A1, SLIT1, SMAD3, SMOX, SNAP23, SPR, STX1A, SYT1, SYT2, SYT5, TAS1R2, TGFB3, TGFBR1, THBS1, TP53, TSC1, TUBA3C/TUBA3D, VAMP2, VEGFA, VTI1B, WASF1, WNT1, WNT3, YKT6, ZDHHC17	ADRA1D, AGTR1, ASIC2, BCL2, BMP3, CACNB3, CACNG8, CASP2, CCND1, CREB1, CREB3L2, CTNNB1, F2RL2, GNAO1, GNAQ, GNAS, HDAC1, HSPA1A, HSPA1B, IKBKE, IL6R, KCNJ8, LEF1, LPAR2, LRRC55, MAP2K1, MAP3K3, MAPK13, MET, MRAS, MYC, MYH9, NOTUM, P2RY2, PIP5K1A, PLA2G2F, PPP1R14D, PPP2R1A, PPP2R3A, PPP2R5A, PRKAB1, PRKCQ, PTPA, RASD2, RHOJ, ROCK1, RRAS, SCNN1G, SMAD3, TGFB3, TGFBR1, THBS1, TP53, VEGFA, WNT1, WNT3
mmu-miR-142-5p	—	Pre-AD	CCNG2, GNG13, IL17F, MYL12A, UBE2D1	ADAM20, CAPN10, FBXL3, GNG13, MYL12A	GNG13, MYL12A
mmu-miR-149	—	Pre-AD	ACVRL1, AQP10, ARHGEF11, ARHGEF18, CACNB2, CLCF1, CRCP, CSF1, CTNNB1, CXCL12, FASLG, FZD5, HSPA9, HSPB7, IL13RA2, IL17RD, IL6, ITGA5, NOTUM, PAK3, PRDX6, RAP1A, RAP1B, RAP2A, RAP2B, SLC9A8, TNFRSF12A	ACVRL1, ADAM12, ADCYAP1R1, ARHGEF11, ARHGEF18, BET1L, BTC, C5AR2, CACNB2, CAPN2, CCR4, CDH26, CDH8, CSNK1G1, CTNNB1, CXCL12, EFNA4, EPHB3, ERBB3, FASLG, FZD5, GCGR, HCAR2, HCAR3, HCK, HSPA9, IL6, IRAK4, ITGA5, KCNJ12, KCNJ3, KCNJ5, LTB4R, NOTUM, PAK3, PAPPA2, PDGFRB, PRDX6, PTGIR, RAP1A, RAP1B, RAP2A, RAP2B, RASGRP2, RGR, SEMA4G, SGTA, SH3GL3, SNCAIP, SNCB, SYK, SYT2, SYT7, TIRAP, VAV2, YKT6	ACVRL1, ARHGEF11, ARHGEF18, CACNB2, CTNNB1, CXCL12, FASLG, FZD5, HSPA9, IL6, ITGA5, NOTUM, PAK3, PRDX6, RAP1A, RAP1B, RAP2A, RAP2B
mmu-miR-200	—	miR-200b Pre-AD miR-200c Pre-AD	ACE2, DNAJB5, EGLN1, ELOC, LPAR1, MAPK7, NOG, PLCG1, PRKACB, PTEN, RAP1B, SMAD5, UBE2W, WNT16	ACE2, CFL2, CRKL, ERBIN, ERRFI1, GABPB1, LPAR1, MAPK7, MARCKS, NRG1, NTF3, PDC, PLCG1, PRKACB, PTEN, PTPN13, RAP1B, SMAD5, SNAP25, TMEFF2, WNT16, ZEB2	ACE2, LPAR1, MAPK7, PLCG1, PRKACB, PTEN, RAP1B, SMAD5, WNT16
mmu-miR-202-5p	—	Pre-AD	CAT, KLK9, MMP20, PDGFD, TEK, TGFBR2	CAT, GRIN2C, HCRTR2, MMP20, NTNG1, PDGFD, RAB9B, TAF9B, TGFBR2, VTI1B	CAT, MMP20, PDGFD, TGFBR2
mmu-miR-382	—	Pre-AD	ANGPTL1, GNG10, IFNG, PKIB, PPP3CC, VIM	ATP5F1C, CAPN8, GNG10, GPR176, IFNA2, IFNA4, IFNG, PPP3CC	GNG10, IFNG, PPP3CC
mmu-miR-423-3p	—	Pre-AD	ABCB8, APOE, ATF6, CACNG6, CAMK2A, CLCF1, CRK, DNAJB12, EIF4EBP1, FGFR2, HNF1A, IL17D, ITGA11, ITGB1, KCNN4, LIF, NGFR, RAC1, RHOBTB1, SMO	APOE, CACNG6, CAMK2A, CREM, CRK, CSPG5, EIF4EBP1, FGFR2, HNF1A, ITGA11, ITGB1, KCNN4, NGFR, NPY4R, RAC1, RHOBTB1, SLC12A5, SMO, TACR3, TAS2R16, THBS2, TUBB6	APOE, CACNG6, CAMK2A, CRK, EIF4EBP1, FGFR2, HNF1A, ITGA11, ITGB1, KCNN4, NGFR, RAC1, RHOBTB1, SMO
mmu-miR-451	—	Pre-AD	ATF2, KLK7, RAC1	ATF2, ATP5F1E, CDH19, GPR34, HCRTR2, PSMB8, RAC1, TSC1, WASF1	ATF2, RAC1
mmu-miR-466g	—	Pre-AD	KLK10, MAP2K4, PPP3CB	CPLX1, EFNA2, GRK4, MAP2K4, PPP3CB, RNF41, TNFRSF11A, UCHL1	MAP2K4, PPP3CB
mmu-miR-467f	—	Pre-AD	AKAP11, ARHGEF10L, ATM, FGF16, FZD7, HSPA14, P2RY1, P2RY12, PIK3C3, PLN, PPP1R14A, PRKCA, PTGS1, TNFRSF1B, UBE2B, UBE2H	ADAMTS2, ADCYAP1, ADGRE4, ARHGEF10L, ASCL1, ATP5MC1, CYSLTR1, FZD7, HLA-DOB, HSPA14, LRRC4C, ORAI2, P2RY1, P2RY12, PIK3C3, PPP1R14A, PRKCA, PTGS1, RAB5B, RAPGEF1, RGS18, RNF41, RPS6KA3, SCN3A, SCN3B, SNAP29, SYT7, TAS2R14, TICAM2	ARHGEF10L, FZD7, HSPA14, P2RY1, P2RY12, PIK3C3, PPP1R14A, PRKCA, PTGS1
mmu-miR-574-3p	—	Pre-AD	EP300, GNAQ, IL6, MMP16, RAC1, TGFBR3	CCKBR, CLTC, EP300, GNAQ, GNRH1, HTR1D, IL6, KLC1, MMP16, NAP1L4, PSMA4, RAC1, RIMS1, SLC6A3, TGFBR3	EP300, GNAQ, IL6, MMP16, RAC1, TGFBR3
mmu-miR-1187	—	Pre-AD	AGTR1, BMP7, CASP6, CASP8, CAT, CCR3, CRCP, DVL3, ENPP1, FGF11, FGF14, FGF23, FOXP3, GNAO1, GNB4, IGF2, IL36G, ITGAM, KCNQ2, MAPK10, MAPK6, MMP12, PLD5, PRKCB, ROCK2, VEGFA	AGTR1, BMP7, CASP6, CASP8, CAT, CCK, CCR3, CCRL2, CPLX2, CYCS, DOK7, GABRB3, GABRG3, GLP1R, GNAO1, GNB4, HRH1, IL36G, ITGAM, KCNQ2, LCK, MAPK10, MAPK6, MMP12, MUSK, NAIP1, NAPB, NLGN3, NRP2, OLIG2, ORAI2, P2RX7, P2RY13, PFN1, PRKCB, RAB7A, RAB9B, RGS1, RGS17, RGS21, RGS4, RGS5, RGS9BP, ROCK2, RORA, RORB, S100B, SEMA5A, SLCO2A1, SNAP23, SPR, SYN3, TACR1, TACR2, TPH1, VEGFA	AGTR1, BMP7, CASP6, CASP8, CAT, CCR3, GNAO1, GNB4, IL36G, ITGAM, KCNQ2, MAPK10, MAPK6, MMP12, PRKCB, ROCK2, VEGFA
mmu-miR-1942	—	Pre-AD	AKAP4, CASP14, EIF2B1, PKIA, PPP2R5A, RHOH	CASP14, PPP2R5A, PSENEN, QRFPR, RHOH, SCN8A	CASP14, PPP2R5A, RHOH
mmu-miR-2183	—	Pre-AD	BMP2	ARPC5L, BMP2, CNR2, HLA-DMB, NECTIN3, POLR2K	BMP2
mmu-miR-125a-5p	—	AD	ABHD3, ALOX5, APLN, APOC4, ARHGEF39, BMPR1B, CASP6, CASP7, DNAJC14, DNAJC19, EPO, HIF1AN, HK2, IL15RA, IL1RN, IL31, IL6R, KLK4, LIF, MAP2K7, MAP3K10, MAP3K11, MAPK12, NFKBIB, PDE7A, PDGFD, PPP1R12B, REL, RHOQ, RHOT2, RPS6KA1, SAA4, SMO, TNFSF4, TP53, UBE2G1, UBE2I, UBE2U	ABHD3, ABHD6, ADAMTS1, ARHGEF39, ARRB1, BMPR1B, CASP6, CASP7, CCR5, CDH5, CNR2, CSNK2A1, ENTPD1, ENTPD4, ERBB2, ERBB3, GPR160, GPR39, H3-3A/H3-3B, HCAR2, HCAR3, ID2, IL1RN, IL6R, LIPA, LRRC8B, MAP2K7, MAP3K10, MAP3K11, MAPK12, MYD88, P2RY8, PDGFD, PPP1R12B, PSMB8, PSMC1, PSME3, PTPN1, REL, RHOQ, RHOT2, RPS6KA1, RXFP4, S1PR3, SCD, SCN2B, SEMA4D, SMO, SSTR3, SYT2, TAF9B, TP53, TRPV1, WAS	ABHD3, ARHGEF39, BMPR1B, CASP6, CASP7, IL1RN, IL6R, MAP2K7, MAP3K10, MAP3K11, MAPK12, PDGFD, PPP1R12B, REL, RHOQ, RHOT2, RPS6KA1, SMO, TP53
mmu-miR-9	—	—	DNAJC14, FGF16, FOXO1, GNA14, HSP90AA1, IFNG, JAK2, KCNMB2, LEP, MYH1, MYOCD, NFKB1, PLA2G2A, RHOA, RHOQ, SHC2, SLC2A2, TNNT2, UTRN	BACE1, CALB2, CDH1, DOK6, FOXO1, GABRB2, GNA14, HLA-F, HSP90AA1, IFNG, JAK1, JAK2, JAK3, KCNJ2, KCNMB2, MTHFD2, MYH1, NAPB, NFKB1, NTF4, NTNG1, NTRK3, PDK4, PLA2G2A, REST, RHOA, RHOQ, SHC2, UTRN, VAV3	FOXO1, GNA14, HSP90AA1, IFNG, JAK2, KCNMB2, MYH1, NFKB1, PLA2G2A, RHOA, RHOQ, SHC2, UTRN
mmu-miR-24	—	—	ABHD3, ACVR1B, APOB, ARHGEF15, BAX, BDKRB1, BIRC5, CCNA2, CXCR2, DNAJB12, DNAJB2, DNAJC16, DUSP1, FASLG, FGF11, HNF1A, HSPB7, IFNG, IL10RB, IL15RA, IL1A, LPAR6, MAP2K4, MAPK14, MAPK7, MYC, NFKBIE, PCYOX1, PDE1A, PPARG, PRKCH, RAP1A, RAP1B, RASD2, SMAD3, SMAD4, SMAD5, TCF7, TNFSF9, TPSAB1, TPSB2, UBE2D4, WNT8B	ABHD3, ACVR1B, ADCYAP1R1, ADORA1, ARHGEF15, ATP5F1D, BAX, BDKRB1, BIRC5, BRCA1, CALCR, CDKN1B, CPLX3, CRH, CRKL, CTSD, ENTPD6, FASLG, GPR151, H3C3, HLA-DRB5, HNF1A, IFNG, IL1A, LAMB3, LPAR6, MAP2K4, MAPK14, MAPK7, MYC, NEFM, NLRP6, NOTCH1, NTSR1, PDE1A, PDGFRB, PER2, PRKCH, PSMB8, RAP1A, RAP1B, RASD2, RNF41, S1PR1, SMAD3, SMAD4, SMAD5, SNCG, SSTR3, TLR8, WNT8B	ABHD3, ACVR1B, ARHGEF15, BAX, BDKRB1, BIRC5, FASLG, HNF1A, IFNG, IL1A, LPAR6, MAP2K4, MAPK14, MAPK7, MYC, PDE1A, PRKCH, RAP1A, RAP1B, RASD2, SMAD3, SMAD4, SMAD5, WNT8B
mmu-miR-26b	—	—	ADM, ATF2, HGF, PRKCD, PTEN, PTGS2, RHOQ, RHOU, SLC9A2, SMAD1, TGFBR2, TPSAB1/TPSB2, UBE2G1, UBE2W	APP, ARPC3, ATF2, BID, BIRC7, CAPN10, EPHA2, GHSR, GPR52, HGF, HTR1A, PRKCD, PTEN, PTGS2, RGS17, RGS4, RHOQ, RHOU, SMAD1, TGFBR2, TRPC3	ATF2, HGF, PRKCD, PTEN, PTGS2, RHOQ, RHOU, SMAD1, TGFBR2
mmu-miR-28	—	—	AGTR2, CACNG1, CDKN1A, FGF23, GNG12, GRK3, IKBKB, MMP15, PDK1, RAP1B, RELA, RHOD, RHOF, WNT5B	AGTR2, CACNG1, CNTFR, DPYSL4, ENTPD5, FSHB, GNG12, GPBAR1, GPR139, GRK3, IKBKB, IL10, IL4I1, KCNJ1, KCNJ6, MMP15, NFE2L2, NPY2R, P2RX1, PDK1, PDK4, PSMF1, PYCARD, RAP1B, RELA, RGS4, RHO, RHOD, RHOF, RPS6KA6, S1PR1, SEMA4C, TAS2R14, TUBB4A, WNT5B	AGTR2, CACNG1, GNG12, GRK3, IKBKB, MMP15, PDK1, RAP1B, RELA, RHOD, RHOF, WNT5B
mmu-miR-30a	—	—	ACTC1, ACVR1, ADRA2A, AKAP14, ATP2A2, BMP5, CASP3, CCN2, CCNA1, COL1A1, COL1A2, CTH, DNAJC13, EDNRA, F2, FZD3, GNA13, GNAI2, GNG10, ITGA2, ITGA8, JUN, MAP3K2, MET, MYO10, NEDD4, NPR3, PDE7A, PDGFA, PDGFB, PIP4K2A, PPP3CA, PPP3R1, PTPA, RAP1B, RASA1, RASD1, SLC4A10, SLC7A1, SMAD1, TCF7, TGFBR1, TGFBR2, TNFSF13B, TP53, UBE2D2, UBE2I, UBE2V2, VIM, WNT5A	ACTC1, ACVR1, ADAMTS14, ADAMTS15, ADAMTS2, ADRA2A, AP2A1, ATP2A2, BMP5, CALB2, CAPN5, CASP3, CTH, CYSLTR1, DOK5, EDNRA, FZD3, GABRB1, GNA13, GNAI2, GNG10, GPR63, HTR1F, ITGA2, ITGA8, JUN, LAMC1, LRRC8C, LRRC8D, MAP3K2, MAP4K4, MET, MICAL1, MYO10, NAPG, NEUROD1, NPR3, PAFAH1B2, PAWR, PDGFA, PDGFB, PFN2, PIP4K2A, PPP3CA, PPP3R1, PTPA, PTPN13, RAP1B, RAPGEF4, RASA1, RASD1, RGS17, RORA, SCN2A, SCN9A, SLC1A2, SLC38A1, SMAD1, STX1A, SYT4, TCF7, TGFBR1, TGFBR2, TP53, VAMP3, VCAN, VIP, WIPF1, WNT5A	ACTC1, ACVR1, ADRA2A, ATP2A2, BMP5, CASP3, CTH, EDNRA, FZD3, GNA13, GNAI2, GNG10, ITGA2, ITGA8, JUN, MAP3K2, MET, MYO10, NPR3, PDGFA, PDGFB, PIP4K2A, PPP3CA, PPP3R1, PTPA, RAP1B, RASA1, RASD1, SMAD1, TCF7, TGFBR1, TGFBR2, TP53, WNT5A
mmu-miR-103	—	—	APLN, AQP11, CACNA2D1, CACNB4, CAV1, CHRNB1, DNAJB2, DNAJB4, FGFRL1, IL10RB, KLK9, NPR3, P2RY12, PDE3B, PDE8B, PIK3CB, PLA2G2D, PNPLA2, PRKAG3, PRKCE, PTEN, PTGS2, RCAN1, TGFBR2, WNT16	ACTR2, ADAM7, BACE1, BDNF, BHLHE40, CACNA2D1, CACNB4, CD80, CDK5R1, CRKL, EPHA3, FGFRL1, GABRA6, GABRB1, GABRG2, GPR183, GRIN2A, HTR2A, HTR4, NDEL1, NPR3, P2RY12, PAWR, PDE3B, PIK3CB, PLA2G2D, PNPLA2, PRKAG3, PRKCE, PTEN, PTGS2, PTH, RAB9B, RET, RGS8, SCN8A, SDCBP, SMPD1, SNCG, STXBP6, SYT2, SYT6, TGFBR2, VCAN, WNT16	CACNA2D1, CACNB4, FGFRL1, NPR3, P2RY12, PDE3B, PIK3CB, PLA2G2D, PNPLA2, PRKAG3, PRKCE, PTEN, PTGS2, TGFBR2, WNT16
mmu-miR-124	—	—	APLN, ARAF, ARHGEF1, BMP6, CAV1, CCL2, CCN2, CHP1, CNTF, CREB3L2, DNAJC1, DNAJC25, DNM2, DVL2, FZD4, GATA6, GNA13, GNAI3, GNG10, GNG2, GRB2, GSK3B, HDAC4, HDAC5, IL11, ITGA7, ITGB1, ITGB3, ITPR3, MAP2K4, MAP3K2, MAPK14, MYH9, MYO10, NFATC1, PGF, PIK3C2A, PIP4K2C, PNPLA2, PPP1R3D, PRKD1, RELA, RHOG, RHOJ, ROCK1, RRAS, RYR1, RYR3, SGK1, SHC1, SMAD5, SNTA1, SNTB2, SP1, STAT3, TF, TNFRSF11B, VIM	ADGRE1, AKT1S1, ALDH9A1, APH1B, ARAF, ARHGEF1, ARPC1B, BDNF, BMP6, CBL, CCL2, CHP1, CNTF, CREB3L2, CTNND1, DNM2, EGR1, FZD4, GLI3, GNA13, GNAI3, GNG10, GNG2, GPR101, GPR119, GRB2, GRIA4, GSK3B, GSN, HDAC4, HDAC5, HLA-DPB1, ITGA7, ITGB1, ITGB3, ITPR3, LAMC1, LMNB1, MAP2K4, MAP3K2, MAPK14, MYH9, MYO10, NEUROD1, NFATC1, NTF4, PDLIM7, PGF, PIK3C2A, PIP4K2C, PNPLA2, PPP1R3D, PRKD1, RAB27A, RASSF5, RELA, RHOG, RHOJ, ROCK1, RRAS, RYR1, RYR3, SGK1, SHC1, SLC16A1, SLCO2A1, SMAD5, SMOX, SNAP23, SOX8, SP1, STAT3, SYT14, TLN1, TUBB4A, TUBB6, VAMP3	ARAF, ARHGEF1, BMP6, CCL2, CHP1, CNTF, CREB3L2, DNM2, FZD4, GNA13, GNAI3, GNG10, GNG2, GRB2, GSK3B, HDAC4, HDAC5, ITGA7, ITGB1, ITGB3, ITPR3, MAP2K4, MAP3K2, MAPK14, MYH9, MYO10, NFATC1, PGF, PIK3C2A, PIP4K2C, PNPLA2, PPP1R3D, PRKD1, RELA, RHOG, RHOJ, ROCK1, RRAS, RYR1, RYR3, SGK1, SHC1, SMAD5, SP1, STAT3
mmu-miR-125b-3p	—	—	CACNG3, DIRAS3, HIF1AN, IL13, IL17RA, IL1B, ORM1, ORM2, PRKCQ, TNF, WNT8B	CACNG3, CPLX2, DIRAS3, DOK3, GNRHR, IL1B, PRKCQ, RGS17, SEMA3B, SERPINF1, SSTR1, TCERG1, TNF, TNFRSF11A, WNT8B	CACNG3, DIRAS3, IL1B, PRKCQ, TNF, WNT8B
mmu-miR-126-5p	—	—	AKAP4, CASP3, CXCL12, F2RL2, FZD3, HSPB8	BTC, CASP3, CXCL12, EREG, F2RL2, FZD3, GABRA6, GPR135, HLA-A, HTR1F, MYD88, POLR2K, SHH, SSTR2	CASP3, CXCL12, F2RL2, FZD3
mmu-miR-128	—	—	ACVR1, APEX1, AQP11, BAX, CACNG2, CAMK1D, CASP8, ECE1, EDNRA, FGF1, GATA6, IL13RA1, IL17B, MAPK14, MMP3, MYL12A, PPP1CC, PRKD1, RAP1B, RND3, RPS6KA5, SP1, TGFBR1, UBE2E2, UBE2N, UBE2W, VEGFC, WNT10B	ACVR1, ADORA2B, AGRN, BAX, CACNG2, CAMK1D, CASP8, CIART, DCX, EDNRA, FGF1, GPR156, GRIN2D, H3-3A, H3-3B, KCNJ6, MAPK14, MMP3, MYL12A, NFIL3, NOTCH1, NOTCH2, NTRK3, PPP1CC, PRKD1, RAP1B, RELN, RGS17, RND3, RPS6KA5, SH2D3C, SLC6A1, SNAP25, SP1, TAAR5, TGFBR1, VEGFC, WNT10B	ACVR1, BAX, CACNG2, CAMK1D, CASP8, EDNRA, FGF1, MAPK14, MMP3, MYL12A, PPP1CC, PRKD1, RAP1B, RND3, RPS6KA5, SP1, TGFBR1, VEGFC, WNT10B
mmu-miR-130b	—	—	ACVR1, BMP5, CALM1, CSF1, CXCL12, EDA, ENPP6, ESR1, FICD, IGF1, IL10RB, MAP3K13, MAPK1, MET, MMP19, PIK3CB, PPARG, PTEN, RASD1, RPS6KA5, SMAD4, SOS2, TGFBR2, UBE2D2, UBE2W, WNT2B	ACVR1, ARHGAP12, BHLHE41, BMP5, CALM1, CSNK1G1, CXCL12, CYSLTR1, DPYSL2, EREG, GABRR1, GJA1, IGF1, KALRN, KCNJ10, KCNJ2, LDAH, LRP8, MAP3K13, MAPK1, MET, NECTIN3, OPN5, PIK3CB, PTEN, PTH, RASD1, RPS6KA5, SMAD4, SNAP25, SOS2, TAC1, TGFBR2, TRPC3, TSC1, UBC, WNT2B	ACVR1, BMP5, CALM1, CXCL12, IGF1, MAP3K13, MAPK1, MET, PIK3CB, PTEN, RASD1, RPS6KA5, SMAD4, SOS2, TGFBR2, WNT2B
mmu-miR-143	—	—	BCL2, BMP5, CAPN3, FGF7, IL18, IL36G, KRAS, MAPK12, MAPK7, MDM2, PLA2G1B, PPP3R2, UBE2E1, UBE2E3, VHL	BCL2, BMP5, CAPN3, GAD2, GPR83, GRIN2B, HLA-DOA, IFNA14, IFNA16, IFNA4, IFNA7, IL18, IL36G, KRAS, MAPK12, MAPK7, MDM2, PLA2G1B, PLXNA2, PPP3R2, XCR1	BCL2, BMP5, CAPN3, IL18, IL36G, KRAS, MAPK12, MAPK7, MDM2, PLA2G1B, PPP3R2
mmu-miR-152	—	—	CSF1, CTH, DNAJC9, DUSP1, EGFR, ESR1, FLT1, IL1RL1, ITGA5, ITGA9, KCNJ8, MAP3K9, MMP10, MMP15, MRAS, NOG, NRAS, PDE1C, PDIA3, PPP1R10, PRKCZ, PTEN, ROCK1, RPS6KA5, SLC2A1, SOS1, SOS2, TBP, TGFA, UBE2D1, UBE2D3, UBE2W, WNT9B	ADAM10, ADAMTS5, ADGRB3, ADGRE1, BHLHE41, CCKBR, CDH7, CDK5R1, CTH, EGFR, ERBB3, ERRFI1, FLT1, GFRA4, GPR183, HCAR1, HLA-A, HLA-B, HLA-C, HLA-DQB2, HOMER1, ITGA5, ITGA9, KCNJ6, KCNJ8, LAMB2, LIPA, LIPG, MAP3K9, MAS1, MMP10, MMP15, MRAS, NRAS, NRP1, P2RY13, PDE1C, PDIA3, POLR2I, PPP1R10, PRKCZ, PTEN, PTGER3, PTPRA, RAB9B, ROBO1, ROCK1, RPS6KA5, RTN4, RXFP1, S1PR1, SLC2A1, SNAP91, SOS1, SOS2, TBP, TGFA, WASL, WNT9B	CTH, EGFR, FLT1, ITGA5, ITGA9, KCNJ8, MAP3K9, MMP10, MMP15, MRAS, NRAS, PDE1C, PDIA3, PPP1R10, PRKCZ, PTEN, ROCK1, RPS6KA5, SLC2A1, SOS1, SOS2, TBP, TGFA, WNT9B
mmu-miR-190	—	—	AGTR1, ANGPTL1, CACNB2, CAT, F2R, FGF10, FGF14, GUCY1A2, GUCY1B1, IL2, P2RY1, PLCZ1, PRKG1, SMAD2, SMPDL3B	ADAM20, ADGRB3, ADGRE3, AGTR1, ARPC1A, ARPC5, CACNB2, CAT, F2R, GPHN, GUCY1A2, GUCY1B1, NEUROD1, NLGN1, P2RY1, PLCZ1, PRKG1, PSMA8, PTGR2, SIGMAR1, SLC17A6, SMAD2, SPRY1	AGTR1, CACNB2, CAT, F2R, GUCY1A2, GUCY1B1, P2RY1, PLCZ1, PRKG1, SMAD2
mmu-miR-191	—	—	CEBPB, FGF20, IL6, MAP3K12, PDE6G, PLCD1, PPP1CB	CRH, CRP, CTTN, EGR1, IL6, MAP3K12, MBP, PDE6G, PLCD1, PPP1CB, TLR3, TUBA1C, UNC5B	IL6, MAP3K12, PDE6G, PLCD1, PPP1CB
mmu-miR-203	—	—	CREB1, FGG, PRKCA	ABL1, CREB1, GPR18, GPR50, H3-3A, H3-3B, KCNJ15, KCNJ6, LRRTM2, PRKCA	CREB1, PRKCA
mmu-miR-342-3p	—	—	BMP7, DKK1, HDAC7, IL3, MYH13, PRKAB2, RAP2A, SCNN1G, UBE2D2	ADAM9, BET1L, BMP7, EREG, HDAC7, HTR1B, HTR2C, ID4, LRP8, MYH13, ORAI1, PRKAB2, RAP2A, RGS4, SCNN1G	BMP7, HDAC7, MYH13, PRKAB2, RAP2A, SCNN1G
mmu-miR-365	—	—	ACVR1, ADM, AQP3, BAX, CDKN1A, CHP1, DOK2, IGF1, IL1A, IL6, ITGAD, KCNH2, LPAR5, MAP3K8, NR3C2, P2RY1, PIK3R3, RASD1, SGK1	ACVR1, ARHGAP12, ARRB2, BAX, CHP1, CYP2C9, DOK2, ENTPD7, HHIP, HTR1F, IGF1, IL1A, IL6, ITGAD, KCNH2, KCNJ2, LPAR5, MAP3K8, P2RY1, PAX6, PIK3R3, RAPGEF4, RASD1, SGK1, TBK1	ACVR1, BAX, CHP1, DOK2, IGF1, IL1A, IL6, ITGAD, KCNH2, LPAR5, MAP3K8, P2RY1, PIK3R3, RASD1, SGK1
mmu-miR-367	—	—	ACTC1, ADCY3, ADRB1, BMPR2, BRAF, CAMK2A, CDKN1A, DNAJB9, DNAJC30, DNAJC4, DSC2, ENPP6, FZD10, GNAQ, HAND1, HAND2, ITGA5, ITGAV, ITGB3, KLF2, MAP2K4, MMP10, NOX4, PDE10A, PIK3R3, PTEN, RAP1B, UBE2W, UBE2Z	ACTC1, ADCY3, ADRB1, BMPR2, BRAF, CAMK2A, CD200R1, DDC, FZD10, GNAQ, GPR180, GRIA1, ITGA5, ITGAV, ITGB3, MAP2K4, MMP10, NEFH, NEFL, NEFM, NOX4, NSF, PIK3R3, PSPN, PTEN, PTGER4, RAB9B, RAP1B, RGS17, RGS3, SLC12A5, SLC17A6, SLC52A1, TAS1R1, WASL, ZEB2	ACTC1, ADCY3, ADRB1, BMPR2, BRAF, CAMK2A, FZD10, GNAQ, ITGA5, ITGAV, ITGB3, MAP2K4, MMP10, NOX4, PIK3R3, PTEN, RAP1B
mmu-miR-433	—	—	ADCY1, F13B, FGF20, GATA3, HDAC6, IL12A, MAPK12, MAPK8, PPP3R1, TFPI, UBE2D2	ADCY1, CD200R1, CYCS, GCH1, HCRTR2, HDAC6, IL12A, MAPK12, MAPK8, OPN5, PPP3R1, SEMA6A, SUCNR1	ADCY1, HDAC6, IL12A, MAPK12, MAPK8, PPP3R1
mmu-miR-434-3p	—	—	BIRC5, HDAC8, NCK1, PLA2G10	BIRC5, HDAC8, NCK1, PAWR, PLA2G10, POLR2K	BIRC5, HDAC8, NCK1, PLA2G10
mmu-miR-486	—	—	CASP3, DNAJC19, FGF7, FOXO1, GLG1, IGF1R, KLK2, LDHA, MAP3K7, MRAS, MYL10, PDGFC, PTEN, RCAN3	CASP3, CCS, CSPG5, EGR2, EPHA5, FOXO1, GLIS1, GPR33, GPR78, IGF1R, MAP3K7, MRAS, MYL10, PDGFC, PTEN, PTGDR, RCOR3, RGS4, S1PR3, SLC38A1, SYT11, VTI1A	CASP3, FOXO1, IGF1R, MAP3K7, MRAS, MYL10, PDGFC, PTEN
mmu-miR-1929	—	—	GNG10, HGF, MMP19, PKIA, PTGS1, TNFSF11	ARPC5L, B2M, CPLX3, DAB1, GFRA4, GNG10, HGF, LNPEP, PTGS1, RGR, RGS8, SDCBP, SMOX	GNG10, HGF, PTGS1

Table 2.

Indicative strength of Alzheimer’s disease pathology and sex differences significance. The list of 230 mRNA targets shared among three or more miRNA regulators has been ordered from the mRNA shared among the most miRNAs on the top (PTEN) to the least towards the bottom. The “Strong”, “Moderate”, “Borderline, “Weak”, and “Absent” descriptors indicate hits among significantly altered miRNAs as 70–100%, 51–69%, = 50%,<50%, and 0% respectively. Cardio, Cardiovascular Signaling pathway string; Neuro, Neurotransmitter and Other Nervous System Signaling pathway string

mRNA	Number of regulating miRNAs	AD Pathology Significance	Biological Sex Significance	Overall Indication Strength
PTEN	Cardio: 11	Cardio: 27%	Cardio: 36%	AD Pathology: Weak
	Neuro: 11	Neuro: 27%	Neuro: 36%	Sex: Weak
GRIN2B	Cardio: N/A	Neuro: 56%	Neuro: 44%	AD Pathology: Moderate
	Neuro: 9			Sex: Weak
RGS17	Cardio: N/A	Neuro: 11%	Neuro: 33%	AD Pathology: Weak
	Neuro: 9			Sex: Weak
TGFBR2	Cardio: 8	Cardio: 25%	Cardio: 25%	AD Pathology: Weak
	Neuro: 8	Neuro: 25%	Neuro: 25%	Sex: Weak
RAP1B	Cardio: 7	Cardio: 0%	Cardio: 29%	AD Pathology: Absent
	Neuro: 7	Neuro: 0%	Neuro: 29%	Sex: Weak
PKIA	Cardio: 7	Cardio: 43%	Cardio: 57%	AD Pathology: Weak
	Neuro: N/A			Sex: Moderate
UBE2D2	Cardio: 7	Cardio: 14%	Cardio: 43%	AD Pathology: Weak
	Neuro: N/A			Sex: Weak
SYT2	Cardio: N/A	Neuro: 29%	Neuro: 71%	AD Pathology: Weak
	Neuro: 7			Sex: Strong
IGF1R	Cardio: 6	Cardio: 50%	Cardio: 67%	AD Pathology: Borderline
	Neuro: 6	Neuro: 50%	Neuro: 67%	Sex: Moderate
MAP2K4	Cardio: 6	Cardio: 17%	Cardio: 33%	AD Pathology: Weak
	Neuro: 6	Neuro: 17%	Neuro: 33%	Sex: Weak
SMAD5	Cardio: 6	Cardio: 50%	Cardio: 33%	AD Pathology: Borderline
	Neuro: 6	Neuro: 50%	Neuro: 33%	Sex: Weak
VEGFA	Cardio: 6	Cardio: 50%	Cardio: 67%	AD Pathology: Borderline
	Neuro: 6	Neuro: 50%	Neuro: 67%	Sex: Moderate
UBE2W	Cardio: 6	Cardio: 0%	Cardio: 17%	AD Pathology: Absent
	Neuro: N/A			Sex: Weak
H3-3A/H3-3B	Cardio: N/A	Neuro: 0%	Neuro: 67%	AD Pathology: Absent
	Neuro: 6			Sex: Moderate
KCNJ6	Cardio: N/A	Neuro: 33%	Neuro: 17%	AD Pathology: Weak
	Neuro: 6			Sex: Weak
LAMC1	Cardio: N/A	Neuro: 33%	Neuro: 33%	AD Pathology: Weak
	Neuro: 6			Sex: Weak
RAB9B	Cardio: N/A	Neuro: 0%	Neuro: 50%	AD Pathology: Absent
	Neuro: 6			Sex: Borderline
RGS4	Cardio: N/A	Neuro: 0%	Neuro: 33%	AD Pathology: Absent
	Neuro: 6			Sex: Weak
RGS8	Cardio: N/A	Neuro: 33%	Neuro: 50%	AD Pathology: Weak
	Neuro: 6			Sex: Borderline
CXCL12	Cardio: 5	Cardio: 40%	Cardio: 40%	AD Pathology: Weak
	Neuro: 5	Neuro: 40%	Neuro: 40%	Sex: Weak
GNG10	Cardio: 5	Cardio: 0%	Cardio: 40%	AD Pathology: Absent
	Neuro: 5	Neuro: 0%	Neuro: 40%	Sex: Weak
IFNG	Cardio: 5	Cardio: 20%	Cardio: 40%	AD Pathology: Weak
	Neuro: 5	Neuro: 20%	Neuro: 40%	Sex: Weak
KRAS	Cardio: 5	Cardio: 40%	Cardio: 40%	AD Pathology: Weak
	Neuro: 5	Neuro: 40%	Neuro: 40%	Sex: Weak
SGK1	Cardio: 5	Cardio: 40%	Cardio: 40%	AD Pathology: Weak
	Neuro: 5	Neuro: 40%	Neuro: 40%	Sex: Weak
SMAD3	Cardio: 5	Cardio: 60%	Cardio: 40%	AD Pathology:
	Neuro: 5	Neuro: 60%	Neuro: 40%	Moderate Sex: Weak
TGFBR1	Cardio: 5	Cardio: 40%	Cardio: 20%	AD Pathology: Weak
	Neuro: 5	Neuro: 40%	Neuro: 20%	Sex: Weak
ARRB1	Cardio: N/A	Neuro: 40%	Neuro: 100%	AD Pathology: Weak
	Neuro: 5			Sex: Strong
CFL2	Cardio: N/A	Neuro: 60%	Neuro: 100%	AD Pathology: Moderate
	Neuro: 5			Sex: Strong
CRKL	Cardio: N/A	Neuro: 20%	Neuro: 40%	AD Pathology: Weak
	Neuro: 5			Sex: Weak
NOTCH1	Cardio: N/A	Neuro: 40%	Neuro: 40%	AD Pathology: Weak
	Neuro: 5			Sex: Weak
NOTCH2	Cardio: N/A	Neuro: 20%	Neuro: 60%	AD Pathology: Weak
	Neuro: 5			Sex: Moderate
PDK4	Cardio: N/A	Neuro: 20%	Neuro: 40%	AD Pathology: Weak
	Neuro: 5			Sex: Weak
PTH	Cardio: N/A	Neuro: 20%	Neuro: 60%	AD Pathology: Weak
	Neuro: 5			Sex: Moderate
SCN8A	Cardio: N/A	Neuro: 20%	Neuro: 60%	AD Pathology: Weak
	Neuro: 5			Sex: Moderate
SLC6A1	Cardio: N/A	Neuro: 60%	Neuro: 60%	AD Pathology: Moderate
	Neuro: 5			Sex: Moderate
SNAP25	Cardio: N/A	Neuro: 40%	Neuro: 40%	AD Pathology: Weak
	Neuro: 5			Sex: Weak
ACVR1	Cardio: 4	Cardio: 0%	Cardio: 0%	AD Pathology: Absent
	Neuro: 4	Neuro: 0%	Neuro: 0%	Sex: Absent
BAX	Cardio: 4	Cardio: 25%	Cardio: 0%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 0%	Sex: Absent
BCL2	Cardio: 4	Cardio: 25%	Cardio: 50%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 50%	Sex: Borderline
CASP3	Cardio: 4	Cardio: 25%	Cardio: 0%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 0%	Sex: Absent
CHP1	Cardio: 4	Cardio: 25%	Cardio: 25%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 25%	Sex: Weak
FOXO1	Cardio: 4	Cardio: 50%	Cardio: 0%	AD Pathology: Borderline
	Neuro: 4	Neuro: 50%	Neuro: 0%	Sex: Absent
FZD3	Cardio: 4	Cardio: 25%	Cardio: 25%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 25%	Sex: Weak
HDAC8	Cardio: 4	Cardio: 25%	Cardio: 75%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 75%	Sex: Strong
IGF1	Cardio: 4	Cardio: 25%	Cardio: 25%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 25%	Sex: Weak
IL36G	Cardio: 4	Cardio: 25%	Cardio: 50%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 50%	Sex: Borderline
IL6	Cardio: 4	Cardio: 0%	Cardio: 50%	AD Pathology: Absent
	Neuro: 4	Neuro: 0%	Neuro: 50%	Sex: Borderline
MAPK7	Cardio: 4	Cardio: 0%	Cardio: 50%	AD Pathology: Absent
	Neuro: 4	Neuro: 0%	Neuro: 50%	Sex: Borderline
MAPK14	Cardio: 4	Cardio: 0%	Cardio: 25%	AD Pathology: Absent
	Neuro: 4	Neuro: 0%	Neuro: 25%	Sex: Weak
MET	Cardio: 4	Cardio: 25%	Cardio: 50%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 50%	Sex: Borderline
MYC	Cardio: 4	Cardio: 25%	Cardio: 50%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 50%	Sex: Borderline
NPR3	Cardio: 4	Cardio: 25%	Cardio: 50%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 50%	Sex: Borderline
P2RY1	Cardio: 4	Cardio: 25%	Cardio: 50%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 50%	Sex: Borderline
PDE1A	Cardio: 4	Cardio: 25%	Cardio: 50%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 50%	Sex: Borderline
PDGFB	Cardio: 4	Cardio: 75%	Cardio: 25%	AD Pathology: Strong
	Neuro: 4	Neuro: 75%	Neuro: 25%	Sex: Weak
PTGS2	Cardio: 4	Cardio: 25%	Cardio: 25%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 25%	Sex: Weak
RAP2A	Cardio: 4	Cardio: 25%	Cardio: 50%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 50%	Sex: Borderline
RPS6KA5	Cardio: 4	Cardio: 25%	Cardio: 0%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 0%	Sex: Absent
SMAD4	Cardio: 4	Cardio: 50%	Cardio: 25%	AD Pathology: Borderline
	Neuro: 4	Neuro: 50%	Neuro: 25%	Sex: Weak
SP1	Cardio: 4	Cardio: 50%	Cardio: 25%	AD Pathology: Borderline
	Neuro: 4	Neuro: 50%	Neuro: 25%	Sex: Weak
TP53	Cardio: 4	Cardio: 25%	Cardio: 50%	AD Pathology: Weak
	Neuro: 4	Neuro: 25%	Neuro: 50%	Sex: Borderline
APLN	Cardio: 4	Cardio: 0%	Cardio: 50%	AD Pathology: Absent
	Neuro: N/A			Sex: Borderline
AQP11	Cardio: 4	Cardio: 25%	Cardio: 25%	AD Pathology: Weak
	Neuro: N/A			Sex: Weak
CDKN1A	Cardio: 4	Cardio: 25%	Cardio: 0%	AD Pathology: Weak
	Neuro: N/A			Sex: Absent
CSF1	Cardio: 4	Cardio: 25%	Cardio: 25%	AD Pathology: Weak
	Neuro: N/A			Sex: Weak
DVL3	Cardio: 4	Cardio: 50%	Cardio: 50%	AD Pathology: Borderline
	Neuro: N/A			Sex: Borderline
ESR1	Cardio: 4	Cardio: 25%	Cardio: 25%	AD Pathology: Weak
	Neuro: N/A			Sex: Weak
FGF7	Cardio: 4	Cardio: 0%	Cardio: 50%	AD Pathology: Absent
	Neuro: N/A			Sex: Borderline
HIF1AN	Cardio: 4	Cardio: 50%	Cardio: 50%	AD Pathology: Borderline
	Neuro: N/A			Sex: Borderline
PPARG	Cardio: 4	Cardio: 25%	Cardio: 25%	AD Pathology: Weak
	Neuro: N/A			Sex: Weak
SNTB2	Cardio: 4	Cardio: 25%	Cardio: 50%	AD Pathology: Weak
	Neuro: N/A			Sex: Borderline
TNFRSF11B	Cardio: 4	Cardio: 75%	Cardio: 25%	AD Pathology: Strong
	Neuro: N/A			Sex: Weak
UBE2D1	Cardio: 4	Cardio: 25%	Cardio: 50%	AD Pathology: Weak
	Neuro: N/A			Sex: Borderline
VIM	Cardio: 4	Cardio: 25%	Cardio: 25%	AD Pathology: Weak
	Neuro: N/A			Sex: Weak
ADAMTS2	Cardio: N/A	Neuro: 50%	Neuro: 25%	AD Pathology: Borderline
	Neuro: 4			Sex: Weak
BACE1	Cardio: N/A	Neuro: 50%	Neuro: 0%	AD Pathology: Borderline
	Neuro: 4			Sex: Absent
CD200R1	Cardio: N/A	Neuro: 25%	Neuro: 25%	AD Pathology: Weak
	Neuro: 4			Sex: Weak
CPLX2	Cardio: N/A	Neuro: 25%	Neuro: 50%	AD Pathology: Weak
	Neuro: 4			Sex: Borderline
CYCS	Cardio: N/A	Neuro: 25%	Neuro: 50%	AD Pathology: Weak
	Neuro: 4			Sex: Borderline
ENTPD7	Cardio: N/A	Neuro: 50%	Neuro: 25%	AD Pathology: Borderline
	Neuro: 4			Sex: Weak
ERBB3	Cardio: N/A	Neuro: 0%	Neuro: 75%	AD Pathology: Absent
	Neuro: 4			Sex: Strong
EREG	Cardio: N/A	Neuro: 25%	Neuro: 25%	AD Pathology: Weak
	Neuro: 4			Sex: Weak
GABRB1	Cardio: N/A	Neuro: 25%	Neuro: 25%	AD Pathology: Weak
	Neuro: 4			Sex: Weak
HLA-A	Cardio: N/A	Neuro: 25%	Neuro: 25%	AD Pathology: Weak
	Neuro: 4			Sex: Weak
KCNJ2	Cardio: N/A	Neuro: 0%	Neuro: 25%	AD Pathology: Absent
	Neuro: 4			Sex: Weak
NTRK3	Cardio: N/A	Neuro: 25%	Neuro: 50%	AD Pathology: Weak
	Neuro: 4			Sex: Borderline
POLR2K	Cardio: N/A	Neuro: 0%	Neuro: 50%	AD Pathology: Absent
	Neuro: 4			Sex: Borderline
RORA	Cardio: N/A	Neuro: 25%	Neuro: 75%	AD Pathology: Weak
	Neuro: 4			Sex: Strong
S1PR1	Cardio: N/A	Neuro: 25%	Neuro: 0%	AD Pathology: Weak
	Neuro: 4			Sex: Absent
S1PR3	Cardio: N/A	Neuro: 25%	Neuro: 50%	AD Pathology: Weak
	Neuro: 4			Sex: Borderline
SCN2B	Cardio: N/A	Neuro: 50%	Neuro: 100%	AD Pathology: Borderline
	Neuro: 4			Sex: Strong
SLC38A1	Cardio: N/A	Neuro: 25%	Neuro: 25%	AD Pathology: Weak
	Neuro: 4			Sex: Weak
SMOX	Cardio: N/A	Neuro: 25%	Neuro: 25%	AD Pathology: Weak
	Neuro: 4			Sex: Weak
SNAP23	Cardio: N/A	Neuro: 25%	Neuro: 50%	AD Pathology: Weak
	Neuro: 4			Sex: Borderline
SYT1	Cardio: N/A	Neuro: 25%	Neuro: 100%	AD Pathology: Weak
	Neuro: 4			Sex: Strong
SYT3	Cardio: N/A	Neuro: 75%	Neuro: 25%	AD Pathology: Strong
	Neuro: 4			Sex: Weak
TSC1	Cardio: N/A	Neuro: 25%	Neuro: 50%	AD Pathology: Weak
	Neuro: 4			Sex: Borderline
VCAN	Cardio: N/A	Neuro: 50%	Neuro: 0%	AD Pathology: Borderline
	Neuro: 4			Sex: Absent
VTI1B	Cardio: N/A	Neuro: 25%	Neuro: 100%	AD Pathology: Weak
	Neuro: 4			Sex: Strong
AGTR1	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 67%	Sex: Moderate
BMP5	Cardio: 3	Cardio: 0%	Cardio: 0%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 0%	Sex: Absent
BMP7	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 67%	Sex: Moderate
CACNG2	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 33%	Sex: Weak
CASP6	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 67%	Sex: Moderate
CASP8	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 33%	Sex: Weak
CAT	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 67%	Sex: Moderate
CCND1	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 67%	Sex: Moderate
CDC42	Cardio: 3	Cardio: 67%	Cardio: 67%	AD Pathology: Moderate
	Neuro: 3	Neuro:67%	Neuro: 67%	Sex: Moderate
CTNNB1	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 67%	Sex: Moderate
EDNRA	Cardio: 3	Cardio: 33%	Cardio: 0%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 0%	Sex: Absent
FASLG	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 33%	Sex: Weak
FGF1	Cardio: 3	Cardio: 67%	Cardio: 33%	AD Pathology: Moderate
	Neuro: 3	Neuro: 67%	Neuro: 33%	Sex: Weak
GNAI3	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 67%	Sex: Moderate
GNAQ	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 67%	Sex: Moderate
GNG12	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 33%	Sex: Weak
GNG5	Cardio: 3	Cardio: 67%	Cardio: 67%	AD Pathology: Moderate
	Neuro: 3	Neuro: 67%	Neuro: 67%	Sex: Moderate
GRB2	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 33%	Sex: Weak
GSK3B	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 33%	Sex: Weak
HGF	Cardio: 3	Cardio: 0%	Cardio: 33%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 33%	Sex: Weak
HMOX1	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 67%	Sex: Moderate
HSPA1A/HSPA1B	Cardio: 3	Cardio: 0%	Cardio: 100%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 100%	Sex: Strong
IL1A	Cardio: 3	Cardio: 33%	Cardio: 0%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 0%	Sex: Absent
IL6R	Cardio: 3	Cardio: 33%	Cardio: 100%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 100%	Sex: Strong
ITGA5	Cardio: 3	Cardio: 0%	Cardio: 33%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 33%	Sex: Weak
ITGB3	Cardio: 3	Cardio: 33%	Cardio: 0%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 0%	Sex: Absent
KCNMB1	Cardio: 3	Cardio: 33%	Cardio: 100%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 100%	Sex: Strong
MAP3K2	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 33%	Sex: Weak
MAPK12	Cardio: 3	Cardio: 0%	Cardio: 33%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 33%	Sex: Weak
MEF2C	Cardio: 3	Cardio: 67%	Cardio: 33%	AD Pathology: Moderate
	Neuro: 3	Neuro: 67%	Neuro: 33%	Sex: Weak
MMP16	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 67%	Sex: Moderate
MRAS	Cardio: 3	Cardio: 0%	Cardio: 33%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 33%	Sex: Weak
MYH1	Cardio: 3	Cardio: 67%	Cardio: 67%	AD Pathology: Moderate
	Neuro: 3	Neuro: 67%	Neuro: 67%	Sex: Moderate
MYL12A	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 33%	Sex: Weak
P2RY12	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 67%	Sex: Moderate
PDGFA	Cardio: 3	Cardio: 67%	Cardio: 0%	AD Pathology: Moderate
	Neuro: 3	Neuro: 67%	Neuro: 0%	Sex: Absent
PDGFD	Cardio: 3	Cardio: 0%	Cardio: 100%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 100%	Sex: Strong
PIK3CB	Cardio: 3	Cardio: 0%	Cardio: 33%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 33%	Sex: Weak
PLA2G2F	Cardio: 3	Cardio: 67%	Cardio: 33%	AD Pathology: Moderate
	Neuro: 3	Neuro: 67%	Neuro: 33%	Sex: Weak
PNPLA2	Cardio: 3	Cardio: 0%	Cardio: 33%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 33%	Sex: Weak
PPP3CA	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 67%	Sex: Moderate
PRKAB2	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 67%	Sex: Moderate
PRKAR2A	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 67%	Sex: Moderate
PRKG1	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 33%	Sex: Weak
PTPA	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 67%	Sex: Moderate
RAC1	Cardio: 3	Cardio: 0%	Cardio: 100%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 100%	Sex: Strong
RASA1	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 67%	Sex: Moderate
RASD1	Cardio: 3	Cardio: 0%	Cardio: 0%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 0%	Sex: Absent
RELA	Cardio: 3	Cardio: 33%	Cardio: 0%	AD Pathology: Weak
	Neuro: 3	Neuro: 33%	Neuro: 0%	Sex: Absent
RHOQ	Cardio: 3	Cardio: 0%	Cardio: 33%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 33%	Sex: Weak
ROCK1	Cardio: 3	Cardio: 0%	Cardio: 33%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 33%	Sex: Weak
WNT2B	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: 3	Neuro: 0%	Neuro: 67%	Sex: Moderate
ATF6	Cardio: 3	Cardio: 0%	Cardio: 100%	AD Pathology: Absent
	Neuro: N/A			Sex: Strong
CCN2	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: N/A			Sex: Weak
CCNA2	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: N/A			Sex: Moderate
CCNG2	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: N/A			Sex: Moderate
CLCF1	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: N/A			Sex: Moderate
COL1A1	Cardio: 3	Cardio: 67%	Cardio: 0%	AD Pathology: Moderate
	Neuro: N/A			Sex: Absent
COL1A2	Cardio: 3	Cardio: 67%	Cardio: 0%	AD Pathology: Moderate
	Neuro: N/A			Sex: Absent
DNAJC13	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: N/A			Sex: Weak
DNAJC14	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: N/A			Sex: Moderate
DNAJC19	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: N/A			Sex: Moderate
DUSP1	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: N/A			Sex: Weak
F2	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: N/A			Sex: Weak
FGF11	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: N/A			Sex: Weak
FGF16	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: N/A			Sex: Moderate
FGF20	Cardio: 3	Cardio: 0%	Cardio: 33%	AD Pathology: Absent
	Neuro: N/A			Sex: Weak
FGF23	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: N/A			Sex: Moderate
GATA6	Cardio: 3	Cardio: 33%	Cardio: 0%	AD Pathology: Weak
	Neuro: N/A			Sex: Absent
IL10RB	Cardio: 3	Cardio: 0%	Cardio: 0%	AD Pathology: Absent
	Neuro: N/A			Sex: Absent
IL11	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: N/A			Sex: Moderate
LTB	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: N/A			Sex: Moderate
NEDD4	Cardio: 3	Cardio: 67%	Cardio: 0%	AD Pathology: Moderate
	Neuro: N/A			Sex: Absent
PDE7A	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: N/A			Sex: Moderate
PDE8B	Cardio: 3	Cardio: 67%	Cardio: 33%	AD Pathology: Moderate
	Neuro: N/A			Sex: Weak
RCAN1	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: N/A			Sex: Moderate
RCAN2	Cardio: 3	Cardio: 67%	Cardio: 33%	AD Pathology: Moderate
	Neuro: N/A			Sex: Weak
TNFSF9	Cardio: 3	Cardio: 33%	Cardio: 33%	AD Pathology: Weak
	Neuro: N/A			Sex: Weak
UBE2G1	Cardio: 3	Cardio: 0%	Cardio: 67%	AD Pathology: Absent
	Neuro: N/A			Sex: Moderate
UBE2V1	Cardio: 3	Cardio: 33%	Cardio: 67%	AD Pathology: Weak
	Neuro: N/A			Sex: Moderate
ADAMTS14	Cardio: N/A	Neuro: 67%	Neuro: 0%	AD Pathology: Moderate
	Neuro: 3			Sex: Absent
ADAMTS15	Cardio: N/A	Neuro: 67%	Neuro: 0%	AD Pathology: Moderate
	Neuro: 3			Sex: Absent
BDNF	Cardio: N/A	Neuro: 0%	Neuro: 33%	AD Pathology: Absent
	Neuro: 3			Sex: Weak
BET1L	Cardio: N/A	Neuro: 33%	Neuro: 33%	AD Pathology: Weak
	Neuro: 3			Sex: Weak
BIRC7	Cardio: N/A	Neuro: 0%	Neuro: 67%	AD Pathology: Absent
	Neuro: 3			Sex: Moderate
BTC	Cardio: N/A	Neuro: 33%	Neuro: 67%	AD Pathology: Weak
	Neuro: 3			Sex: Moderate
CCK	Cardio: N/A	Neuro: 33%	Neuro: 100%	AD Pathology: Weak
	Neuro: 3			Sex: Strong
CD80	Cardio: N/A	Neuro: 0%	Neuro: 67%	AD Pathology: Absent
	Neuro: 3			Sex: Moderate
CDK5R1	Cardio: N/A	Neuro: 0%	Neuro: 33%	AD Pathology: Absent
	Neuro: 3			Sex: Weak
CDKN1B	Cardio: N/A	Neuro: 33%	Neuro: 33%	AD Pathology: Weak
	Neuro: 3			Sex: Weak
CRP	Cardio: N/A	Neuro: 33%	Neuro: 33%	AD Pathology: Weak
	Neuro: 3			Sex: Weak
CSF1R	Cardio: N/A	Neuro: 33%	Neuro: 100%	AD Pathology: Weak
	Neuro: 3			Sex: Strong
CXCL10	Cardio: N/A	Neuro: 33%	Neuro: 67%	AD Pathology: Weak
	Neuro: 3			Sex: Moderate
CYSLTR1	Cardio: N/A	Neuro: 0%	Neuro: 33%	AD Pathology: Absent
	Neuro: 3			Sex: Weak
EGR1	Cardio: N/A	Neuro: 0%	Neuro: 33%	AD Pathology: Absent
	Neuro: 3			Sex: Weak
FSHB	Cardio: N/A	Neuro: 33%	Neuro: 33%	AD Pathology: Weak
	Neuro: 3			Sex: Weak
GABRA6	Cardio: N/A	Neuro: 33%	Neuro: 33%	AD Pathology: Weak
	Neuro: 3			Sex: Weak
GCH1	Cardio: N/A	Neuro: 33%	Neuro: 67%	AD Pathology: Weak
	Neuro: 3			Sex: Moderate
GPR101	Cardio: N/A	Neuro: 0%	Neuro: 67%	AD Pathology: Absent
	Neuro: 3			Sex: Moderate
GPR183	Cardio: N/A	Neuro: 0%	Neuro: 33%	AD Pathology: Absent
	Neuro: 3			Sex: Weak
GPR55	Cardio: N/A	Neuro: 67%	Neuro: 33%	AD Pathology: Moderate
	Neuro: 3			Sex: Weak
GPR63	Cardio: N/A	Neuro: 33%	Neuro: 33%	AD Pathology: Weak
	Neuro: 3			Sex: Weak
HCRTR2	Cardio: N/A	Neuro: 0%	Neuro: 67%	AD Pathology: Absent
	Neuro: 3			Sex: Moderate
HOMER1	Cardio: N/A	Neuro: 0%	Neuro: 67%	AD Pathology: Absent
	Neuro: 3			Sex: Moderate
HTR1F	Cardio: N/A	Neuro: 0%	Neuro: 0%	AD Pathology: Absent
	Neuro: 3			Sex: Absent
IFNA4	Cardio: N/A	Neuro: 33%	Neuro: 33%	AD Pathology: Weak
	Neuro: 3			Sex: Weak
IL10	Cardio: N/A	Neuro: 33%	Neuro: 33%	AD Pathology: Weak
	Neuro: 3			Sex: Weak
KCNJ1	Cardio: N/A	Neuro: 67%	Neuro: 0%	AD Pathology: Moderate
	Neuro: 3			Sex: Absent
LRP8	Cardio: N/A	Neuro: 0%	Neuro: 33%	AD Pathology: Absent
	Neuro: 3			Sex: Weak
NAPB	Cardio: N/A	Neuro: 33%	Neuro: 33%	AD Pathology: Weak
	Neuro: 3			Sex: Weak
NAPG	Cardio: N/A	Neuro: 0%	Neuro: 67%	AD Pathology: Absent
	Neuro: 3			Sex: Moderate
NEFM	Cardio: N/A	Neuro: 33%	Neuro: 0%	AD Pathology: Weak
	Neuro: 3			Sex: Absent
NEUROD1	Cardio: N/A	Neuro: 0%	Neuro: 0%	AD Pathology: Absent
	Neuro: 3			Sex: Absent
NFE2L2	Cardio: N/A	Neuro: 33%	Neuro: 67%	AD Pathology: Weak
	Neuro: 3			Sex: Moderate
NTNG1	Cardio: N/A	Neuro: 33%	Neuro: 33%	AD Pathology: Weak
	Neuro: 3			Sex: Weak
OPN5	Cardio: N/A	Neuro: 33%	Neuro: 0%	AD Pathology: Weak
	Neuro: 3			Sex: Absent
ORAI2	Cardio: N/A	Neuro: 0%	Neuro: 100%	AD Pathology: Absent
	Neuro: 3			Sex: Strong
PAPPA	Cardio: N/A	Neuro: 67%	Neuro: 67%	AD Pathology: Moderate
	Neuro: 3			Sex: Moderate
PAWR	Cardio: N/A	Neuro: 0%	Neuro: 0%	AD Pathology: Absent
	Neuro: 3			Sex: Absent
PER2	Cardio: N/A	Neuro: 33%	Neuro: 67%	AD Pathology: Weak
	Neuro: 3			Sex: Moderate
POLR2D	Cardio: N/A	Neuro: 67%	Neuro: 67%	AD Pathology: Moderate
	Neuro: 3			Sex: Moderate
PSMB8	Cardio: N/A	Neuro: 0%	Neuro: 67%	AD Pathology: Absent
	Neuro: 3			Sex: Moderate
RGS1	Cardio: N/A	Neuro: 67%	Neuro: 33%	AD Pathology: Moderate
	Neuro: 3			Sex: Weak
RNF41	Cardio: N/A	Neuro: 0%	Neuro: 67%	AD Pathology: Absent
	Neuro: 3			Sex: Moderate
SCN2A	Cardio: N/A	Neuro: 0%	Neuro: 67%	AD Pathology: Absent
	Neuro: 3			Sex: Moderate
SIRT1	Cardio: N/A	Neuro: 33%	Neuro: 100%	AD Pathology: Weak
	Neuro: 3			Sex: Strong
SNCG	Cardio: N/A	Neuro: 0%	Neuro: 33%	AD Pathology: Absent
	Neuro: 3			Sex: Weak
SYT4	Cardio: N/A	Neuro: 33%	Neuro: 67%	AD Pathology: Weak
	Neuro: 3			Sex: Moderate
TAF9B	Cardio: N/A	Neuro: 33%	Neuro: 67%	AD Pathology: Weak
	Neuro: 3			Sex: Moderate
TUBB4A	Cardio: N/A	Neuro: 33%	Neuro: 0%	AD Pathology: Weak
	Neuro: 3			Sex: Absent
VAMP2	Cardio: N/A	Neuro: 67%	Neuro: 67%	AD Pathology: Moderate
	Neuro: 3			Sex: Moderate
XCR1	Cardio: N/A	Neuro: 33%	Neuro: 33%	AD Pathology: Weak
	Neuro: 3			Sex: Weak

Table 3.

mRNA targets that indicate development of Alzheimer’s disease (AD) pathology. Only mRNA targets shared among at least three miRNAs are shown. Significantly altered miRNAs during the course of AD pathology include: mmu-let-7d, mmu-miR-181a, mmu-miR-132, mmu-miR-99, mmu-miR-151-5p, mmu-miR-23, mmu-miR-126-3p, mmu-miR-150, mmu-miR-27a, mmu-miR-135a, mmu-miR-133a, mmu-miR-690, mmu-miR-29c, mmu-miR-34b-3p, mmu-miR-129-3p, and mmu-miR-539. Cardio, Cardiovascular Signaling pathway string; Neuro, Neurotransmitter and Other Nervous System Signaling pathway string

mRNA, pathway and # of regulating miRNAs	Regulating miRNAs	AD pathology Significance by % of changing miRNA hits out of total miRNAs per mRNA	Function
PDGFB, Cardio and Neuro (4 miRNAs)	mmu-let-7d, mmu-miR-150, mmu-miR-29c, mmu-miR-30a	Strong (75%)	Platelet-derived growth factor; pericyte/smooth muscle cell recruitment, angiogenesis
TNFRSF11B, Cardio (4 miRNAs)	mmu-miR-124, mmu-miR-132, mmu-miR-135a, mmu-miR-181a	Strong (75%)	Tumor necrosis factor receptor member; inhibition of osteoclast activation, osteoclast apoptosis
SYT3, Neuro (4 miRNAs)	mmu-miR-135a, mmu-miR-151-5p, mmu-miR-16, mmu-miR-27a	Strong (75%)	Synaptotagmin; Ca2+-dependent exocytosis and syntaxin binding
GRIN2B, Neuro (9 miRNAs)	mmu-let-7d, mmu-miR-129-3p, mmu-miR-135a, mmu-miR-143, mmu-miR-150, mmu-miR-181a, mmu-miR-204, mmu-miR-34c, mmu-miR-377	Moderate (56%)	NMDA receptor subunit; Synaptic plasticity, cerebral blood flow regulation
SMAD3, Cardio and Neuro (5 miRNAs)	mmu-miR-129-3p, mmu-miR-23b, mmu-miR-24, mmu-miR-27a, mmu-miR-34c	Moderate (60%)	Transforming growth factor β-signaling molecule; cell proliferation, tumor suppression
CFL2, Neuro (5 miRNAs)	mmu-miR-132, mmu-miR-16, mmu-miR-200c, mmu-miR-23b, mmu-miR-34b-3p	Moderate (60%)	Component of intranuclear and cytoplasmic actin rods; cytoskeletal development and regulation
SLC6A1, Neuro (5 miRNAs)	mmu-miR-128, mmu-miR-132, mmu-miR-133a, mmu-miR-27a, mmu-miR-34c	Moderate (60%)	GABA transporter; reuptake and termination of GABA activity
CDC42, Cardio and Neuro (3 miRNAs)	mmu-miR-133a, mmu-miR-29c, mmu-miR-16	Moderate (67%)	Cell division cycle 42; GTP-dependent protein binding and GTPase activity, dendritic spine morphogenesis, heart development
FGF1, Cardio and Neuro (3 miRNAs)	mmu-miR-128, mmu-miR-133a mmu-miR-27a	Moderate (67%)	Fibroblast Growth Factor member; cell division/differentiation/migration and angiogenesis
GNG5, Cardio and Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-34b-3p, mmu-miR-377	Moderate (67%)	G protein subunit gamma 5; anti-inflammatory cytokine production, thromboxane signaling
MEF2C, Cardio and Neuro (3 miRNAs)	mmu-miR-135a, mmu-miR-204, mmu-miR-27a	Moderate (67%)	Myocyte enhancer factor 2C; vascular development, cardiac morphogenesis
MYH1, Cardio and Neuro (3 miRNAs)	mmu-miR-150, mmu-miR-23b, mmu-miR-9	Moderate (67%)	Myosin heavy chain 1; actin binding, vascular contractility
PDGFA, Cardio and Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-29c, mmu-miR-30a	Moderate (67%)	Platelet-derived growth factor; angiogenesis, cell proliferation and differentiation
PLA2G2F, Cardio and Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-129-3p, mmu-miR-34c	Moderate (67%)	Phospholipase A2, group IIF; Ca2+-dependent phospholipase activity
COL1A1, Cardio (3 miRNAs)	mmu-let-7d, mmu-miR-29c, mmu-miR-30a	Moderate (67%)	Matrisome member; Collagen formation
COL1A2, Cardio (3 miRNAs)	mmu-let-7d, mmu-miR-29c, mmu-miR-30a	Moderate (67%)	Matrisome member Collagen formation
NEDD4, Cardio (3 miRNAs)	mmu-let-7d, mmu-miR-27a, mmu-miR-30a	Moderate (67%)	Ubiquitin proteasome member; degradation of membrane receptors, endocytic machinery components, and tumor suppressor PTEN
PDE8B, Cardio (3 miRNAs)	mmu-miR-103, mmu-miR-133a, mmu-miR-135a	Moderate (67%)	Cyclic nucleotide phosphodiesterase; hydrolyzes second messenger cAMP
RCAN2, Cardio (3 miRNAs)	mmu-miR-129-3p, mmu-miR-27a, mmu-miR-377	Moderate (67%)	Regulator of calcineurin 2; enables Ca2+-dependent protein serine/threonine phosphatase regulator activity
ADAMTS14, Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-29c, mmu-miR-30a	Moderate (67%)	Disintegrin/metalloprotease; amino procollagen type I processing in absence of ADAMTS2
ADAMTS15, Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-29c, mmu-miR-30a	Moderate (67%)	Disintegrin/metalloprotease; proteolytic activity against the proteoglycan VCAN
GPR55, Neuro (3 miRNAs)	mmu-miR-129-3p, mmu-miR-135a, mmu-miR-22	Moderate (67%)	G protein-coupled receptor 55; cannabinoid receptor activity, activation of phospholipase C activity, bone resorption, and positive regulation of intracellular signal transduction
KCNJ1, Neuro (3 miRNAs)	mmu-miR-126-3p, mmu-miR-129-3p, mmu-miR-28	Moderate (67%)	Inward Rectifying K+ Channel; K+ homeostasis and membrane potential regulation
PAPPA, Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-133a, mmu-miR-16	Moderate (67%)	Pregnancy-associated plasma protein; endopeptidase activity, proteolysis
POLR2D, Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-132, mmu-miR-146a	Moderate (67%)	Polymerase (RNA) II (DNA directed) polypeptide D; translation initiation factor binding activity, RNA metabolic processes, mRNA export from nucleus in response to heart stress, positive regulation of translational initiation
RGS1, Neuro (3 miRNAs)	mmu-miR-27a, mmu-miR-29c, mmu-miR-1187	Moderate (67%)	Regulator of G-protein signaling 1; G-protein alpha-subunit binding activity and GTPase activator activity
VAMP2, Neuro (3 miRNAs)	mmu-miR-133a, mmu-miR-151-5p, mmu-miR-34c	Moderate (67%)	Vesicle-associated membrane protein 2; calmodulin binding activity, phospholipid activity, and syntaxin-1 binding activity, and is involved in secretion by cell and synaptic vesicle endocytosis
IGF1R, Cardio and Neuro (6 miRNAs)	mmu-let-7d, mmu-miR-99b, mmu-miR-133a, mmu-miR-145, mmu-miR-16, mmu-miR-486	Borderline (50%)	Insulin-like growth factor I receptor; insulin-like growth factor binding activity, adrenal gland development, positive regulation of cold-induced thermogenesis, positive regulation of meiotic cell cycle
SMAD5, Cardio and Neuro (6 miRNAs)	mmu-miR-124, mmu-miR-135a, mmu-miR-200c, mmu-miR-23b, mmu-miR-24, mmu-miR-27a	Borderline (50%)	Transforming growth factor β-signaling molecule; inhibition of hematopoietic progenitor cell proliferation
VEGFA, Cardio and Neuro (6 miRNAs)	mmu-miR-126-3p, mmu-miR-150, mmu-miR-16, mmu-miR-29c, mmu-miR-34c, mmu-miR-1187	Borderline (50%)	Vascular endothelial growth factor; endothelial cell growth, angiogenesis, vasculogenesis
FOXO1, Cardio and Neuro (4 miRNAs)	mmu-miR-135a, mmu-miR-27a, mmu-miR-486, mmu-miR-9	Borderline (50%)	Forkhead transcription factor; insulin signaling, metabolic homeostasis
SMAD4, Cardio and Neuro (4 miRNAs)	mmu-miR-130b, mmu-miR-23b, mmu-miR-24, mmu-miR-27a	Borderline (50%)	Transforming growth factor β-signaling molecule; cell growth and proliferation
SP1, Cardio and Neuro (4 miRNAs)	mmu-miR-124, mmu-miR-128, mmu-miR-150, mmu-miR-29c	Borderline (50%)	Trans-acting transcription factor 1; regulation of transcription, DNA-templated and response to hydroperoxide
DVL3, Cardio (4 miRNAs)	mmu-let-7d, mmu-miR-129-3p, mmu-miR-204, mmu-miR-1187	Borderline (50%)	Disheveled segment polarity protein 3; protein-macromolecule adaptor activity, canonical and non-canonical Wnt signaling pathway via JNK cascade
HIF1AN, Cardio (4 miRNAs)	mmu-let-7d, mmu-miR-125b-3p, mmu-miR-125a-5p, mmu-miR-23b	Borderline (50%)	Hypoxia-inducible factor 1, alpha subunit inhibitor; 2-oxoglutarate-dependent dioxygenase activity, NF-kappaB binding activity, transition metal binding activity
ADAMTS2, Neuro (4 miRNAs)	mmu-let-7d, mmu-miR-29c, mmu-miR-30a, mmu-miR-467f	Borderline (50%)	Disintegrin/metalloprotease; cleavage of type I/II collagen propeptides
BACE1, Neuro (4 miRNAs)	mmu-miR-103, mmu-miR-129-3p, mmu-miR-29c, mmu-miR-9	Borderline (50%)	Beta-site APP cleaving enzyme 1; catalyzes first step in formation of amyloid-β peptide from amyloid precursor protein
ENTPD7, Neuro (4 miRNAs)	mmu-miR-135a, mmu-miR-16, mmu-miR-29c, mmu-miR-365	Borderline (50%)	Ectonucleoside triphosphate diphosphohydrolase 7; nucleoside-triphosphatase activity
SCN2B, Neuro (4 miRNAs)	mmu-miR-125a-5p, mmu-miR-133a, mmu-miR-150, mmu-miR-34c	Borderline (50%)	Na+ channel, voltage-gated, type II, beta; Na+ channel regulator activity in cardiac muscle and cellular action potentials
VCAN, Neuro (4 miRNAs)	mmu-let-7d, mmu-miR-103, mmu-miR-29c, mmu-miR-30a	Borderline (50%)	Versican; protein phosphatase binding activity

Table 4.

mRNA targets that indicate significant expression differences among cerebrovascular miRNAs of males and females of Pre-AD and/or AD groups. Only mRNA targets shared among at least three miRNAs are shown. Significantly different miRNAs among males and females include: mmu-miR-132, mmu-miR-99, mmu-miR-23, mmu-miR-150, mmu-miR-133a, mmu-miR-34b-3p, mmu-miR-539, mmu-miR-145, mmu-miR-146a, mmu-miR-377, mmu-miR-101b, mmu-miR-204, mmu-miR-16, mmu-miR-22, mmu-miR-34c, mmu-miR-142-5p, mmu-miR-149, mmu-miR-200, mmu-miR-202-5p, mmu-miR-382, mmu-miR-423-3p, mmu-miR-451, mmu-miR-466g, mmu-miR-467f, mmu-miR-574-3p, mmu-miR-1187, mmu-miR-1942, mmu-miR-2183, mmu-miR-125a-5p. Cardio, Cardiovascular Signaling pathway string; Neuro, Neurotransmitter and Other Nervous System Signaling pathway string

mRNA/protein, pathway and # of regulating miRNAs	Regulating miRNAs	Biological Sex Significance by % of changing miRNA hits out of total miRNAs per mRNA	Function
SYT2, Neuro (7 miRNAs)	mmu-miR-103, mmu-miR-125a-5p, mmu-miR-133a, mmu-miR-135a, mmu-miR-149, mmu-miR-34c, mmu-miR-377	Strong (71%)	Synaptotagmin; Ca2+ ion binding and dendrite formation
ARRB1, Neuro (5 miRNAs)	mmu-miR-125a-5p, mmu-miR-133a, mmu-miR-150, mmu-miR-22, mmu-miR-377	Strong (100%)	Arrestin, beta 1; cysteine-type endopeptidase inhibitor activity, apoptotic processes and mitogen-activated protein kinase binding activity
CFL2, Neuro (5 miRNAs)	mmu-miR-132, mmu-miR-16, mmu-miR-200c, mmu-miR-23b, mmu-miR-34b-3p	Strong (100%)	Component of intranuclear and cytoplasmic actin rods; cytoskeletal development and regulation
HDAC8, Cardio and Neuro (4 miRNAs)	mmu-miR-146a, mmu-miR-150, mmu-miR-377, mmu-miR-434-3p	Strong (75%)	Histone deacetylase related to sirtuins; transcriptional, cell cycle, and developmental regulation
ERBB3, Neuro (4 miRNAs)	mmu-miR-125a-5p, mmu-miR-152, mmu-miR-149, mmu-miR-22	Strong (75%)	Receptor tyrosine kinase of the EGFR family; cell proliferation and differentiation
RORA, Neuro (4 miRNAs)	mmu-miR-99b, mmu-miR-30a, mmu-miR-377, mmu-miR-1187	Strong (75%)	Related orphan receptor alpha; binds as a monomer or as a homodimer to hormone response elements upstream of several genes to enhance gene expression
SYT1, Neuro (4 miRNAs)	mmu-miR-133a, mmu-miR-146a, mmu-miR-34c, mmu-miR-377	Strong (100%)	Synaptotagmin; Ca2+ ion binding and neurotransmission
VTI1B, Neuro (4 miRNAs)	mmu-miR-16, mmu-miR-202-5p, mmu-miR-23b, mmu-miR-34c	Strong (100%)	Vesicle transport through interaction with t-SNAREs 1B; SNAP receptor activity, SNARE binding activity, and Cl− channel inhibitor activity
HSPA1A/1B, Cardio and Neuro (3 miRNAs)	mmu-miR-146a, mmu-miR-16, mmu-miR-34c	Strong (100%)	Heat shock protein 70 family member; molecular chaperone and proteome protection
IL6R, Cardio and Neuro (3 miRNAs)	mmu-miR-125a-5p, mmu-miR-23b, mmu-miR-34c	Strong (100%)	Interleukin 6 receptor; interleukin-6 binding and receptor activity
KCNMB1, Cardio and Neuro (3 miRNAs)	mmu-miR-133a, mmu-miR-16, mmu-miR-22	Strong (100%)	Large conductance Ca2+-activated K+ channel; Ca2+-activated K+ channel activity, vasodilation
PDGFD, Cardio and Neuro (3 miRNAs)	mmu-miR-125a-5p, mmu-miR-145, mmu-miR-202-5p	Strong (100%)	Platelet-derived growth factor, D polypeptide; platelet-derived growth factor binding activity
RAC1, Cardio and Neuro (3 miRNAs)	mmu-miR-423-3p, mmu-miR-451, mmu-miR-574-3p	Strong (100%)	Rac family small GTPase1; GTP binding activity, GTP-dependent protein binding activity, and GTPase activity
ATF6, Cardio (3 miRNAs)	mmu-miR-146a, mmu-miR-16, mmu-miR-423-3p	Strong (100%)	Activation transcription factor 6; DNA-binding transcription activator activity, RNA-polymerase II-specific, protein heterodimerization activity, and ubiquitin protein ligase binding activity
CCK, Neuro (3 miRNAs)	mmu-miR-146a, mmu-miR-23b, mmu-miR-1187	Strong (100%)	Cholecystokinin; encodes a member of the gastrin/cholecystokinin family of proteins to generate protein products to regulate gastric acid secretion and food intake
CSF1R, Neuro (3 miRNAs)	mmu-miR-150, mmu-miR-22, mmu-miR-34c	Strong (100%)	Colony stimulating factor 1 receptor; macrophage colony-stimulating factor receptor activity, protein homodimerization activity, and protein phosphatase binding activity
ORAI2, Neuro (3 miRNAs)	mmu-miR-22, mmu-miR-1187, mmu-miR-467f	Strong (100%)	ORAI Ca2+ release-activated Ca2+ modulator 2; store-operated Ca2+ channel activity
SCN2B, Neuro (4 miRNAs)	mmu-miR-125a-5p, mmu-miR-133a, mmu-miR-150, mmu-miR-34c	Strong (100%)	Na+ channel, voltage-gated, type II, beta; Na+ channel activity in cardiac muscle cell action potentials
SIRT1, Neuro (3 miRNAs)	mmu-miR-132, mmu-miR-22, mmu-miR-34c	Strong (100%)	Sirtuin 1; proposed role in longevity by regulating gene expression through its NAD+ dependent deacetylation of histones, transcription factors, and transcriptional coactivators
PKIA, Cardio (7 miRNAs)	mmu-miR-129-3p, mmu-miR-1929, mmu-miR-1942, mmu-miR-23b, mmu-miR-27a, mmu-miR-34c, mmu-miR-377	Moderate (57%)	Protein kinase inhibitor, alpha; cAMP-dependent protein kinase inhibitor activity and protein kinase A catalytic subunit binding activity
H3-3A/H3-3B, Neuro (6 miRNAs)	mmu-miR-125a-5p, mmu-miR-128, mmu-miR-16, mmu-miR-203, mmu-miR-22, mmu-miR-377	Moderate (67%)	H3.3 histone A/H3.3 histone B; nucleosome structure of the chromosomal fiber in eukaryotes
NOTCH2, Neuro (5 miRNAs)	mmu-miR-128, mmu-miR-16, mmu-miR-27a, mmu-miR-34c, mmu-miR-377	Moderate (60%)	Notch 2; NF-kappaB binding activity and enzyme binding activity
PTH, Neuro (5 miRNAs)	mmu-miR-101b, mmu-miR-103, mmu-miR-130b, mmu-miR-16, mmu-miR-539	Moderate (60%)	Parathyroid hormone; blood Ca2+ and phosphate level regulation
SCN8A, Neuro (5 miRNAs)	mmu-let-7d, mmu-miR-103, mmu-miR-16, mmu-miR-1942, mmu-miR-377	Moderate (60%)	Na+ channel, voltage-gated, type VIII; Na+ ion binding activity and Na+ channel activity
SLC6A1, Neuro (5 miRNAs)	mmu-miR-128, mmu-miR-132, mmu-miR-133a, mmu-miR-27a, mmu-miR-34c	Moderate (60%)	GABA transporter; reuptake and termination of GABA activity
GNG5, Cardio and Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-34b-3p, mmu-miR-377	Moderate (67%)	G protein subunit gamma 5; G-protein beta subunit binding activity and PDZ domain binding activity
MYH1, Cardio and Neuro (3 miRNAs)	mmu-miR-150, mmu-miR-23b, mmu-miR-9	Moderate (67%)	Myosin, heavy polypeptide 1, skeletal muscle; ATP binding activity, actin filament binding activity, and calmodulin binding activity
AGTR1, Cardio and Neuro (3 miRNAs)	mmu-miR-190, mmu-miR-34c, mmu-miR-1187	Moderate (67%)	Angiotensin II receptor; positive regulation of cytokine production and regulation of blood pressure
BMP7, Cardio and Neuro (3 miRNAs)	mmu-miR-22, mmu-miR-342-3p, mmu-miR-1187	Moderate (67%)	Bone morphogenetic protein 7; encodes a secreted ligand of the TGF-beta superfamily of proteins
CASP6, Cardio and Neuro (3 miRNAs)	mmu-miR-125a-5p, mmu-miR-129-3, mmu-miR-1187	Moderate (67%)	Caspase 6; transmission of pain and axonal degeneration
CAT, Cardio and Neuro (3 miRNAs)	mmu-miR-190 mmu-miR-202-5p mmu-miR-1187	Moderate (67%)	Catalase; aminoacylase activity and catalase activity
CCND1, Cardio and Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-16, mmu-miR-34c	Moderate (67%)	Cyclin; cell-cycle regulation during G1/S transition
CDC42, Cardio and Neuro (3 miRNAs)	mmu-miR-133a, mmu-miR-29c, mmu-miR-16	Moderate (67%)	Cell division cycle 42; GTP-dependent protein binding and GTPase activity, dendritic spine morphogenesis, heart development
CTNNB1, Cardio and Neuro (3 miRNAs)	mmu-miR-149, mmu-miR-34c, mmu-miR-690	Moderate (67%)	Cadherin associated protein, beta 1; adhesive function of classical cadherins and mediates the canonical Wnt signaling pathway for gene expression
GNAI3, Cardio and Neuro (3 miRNAs)	mmu-miR-124, mmu-miR-16, mmu-miR-22	Moderate (67%)	Gene protein-coupled receptor α-subunit; receptor-regulated K+ channel, transmembrane signaling
GNAQ, Cardio and Neuro (3 miRNAs)	mmu-miR-34c, mmu-miR-574-3p, mmu-miR-367	Moderate (67%)	Guanine nucleotide binding protein, alpha q polypeptide; G-protein beta/gamma-subunit complex binding activity, GTPase activator activity, and alkyl glycerophosphoethanolamine phosphodiesterase activity
HMOX1, Cardio and Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-16, mmu-miR-377	Moderate (67%)	Heme oxygenase; heme degradation and carbon monoxide production
PAPPA, Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-133a, mmu-miR-16	Moderate (67%)	Pregnancy-associated plasma protein; endopeptidase activity
MMP16, Cardio and Neuro (3 miRNAs)	mmu-miR-129-3p, mmu-miR-146a, mmu-miR-574-3p	Moderate (67%)	Matrix metalloproteinase; degradation of collagen and fibronectin
POLR2D, Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-132, mmu-miR-146a	Moderate (67%)	Polymerase (RNA) II (DNA directed) polypeptide D; translation initiation factor binding activity, RNA metabolic process, mRNA export from nucleus in response to heat stress, and positive regulation of translation initiation
P2RY12, Cardio and Neuro (3 miRNAs)	mmu-miR-101b, mmu-miR-103, mmu-miR-467f	Moderate (67%)	Purinergic receptor P2Y, G-protein coupled 12; G-protein-coupled ADP receptor activity and G protein-coupled adenosine receptor activity
VAMP2, Neuro (3 miRNAs)	mmu-miR-133a, mmu-miR-151-5p, mmu-miR-34c	Moderate (67%)	Vesicle-associated membrane protein; calmodulin binding activity, phospholipid binding activity, and syntaxin-1 binding activity
PPP3CA, Cardio and Neuro (3 miRNAs)	mmu-miR-99b, mmu-miR-145, mmu-miR-30a	Moderate (67%)	Protein Phosphatase 3 Catalytic Subunit α; osteoclast differentiation and immunity
PRKAB2, Cardio and Neuro (3 miRNAs)	mmu-miR-204, mmu-miR-342-3p, mmu-miR-377	Moderate (67%)	Protein kinase, AMP-activated, beta 2 non-catalytic subunit; AMP-activated protein kinase activity, positive regulation of cold-induced thermogenesis
PRKAR2A, Cardio and Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-16, mmu-miR-22	Moderate (67%)	Protein kinase, cAMP dependent regulatory, type II alpha; modulation of chemical synaptic transmission
PTPA, Cardio and Neuro (3 miRNAs)	mmu-miR-132, mmu-miR-30a, mmu-miR-34c	Moderate (67%)	Protein phosphatase 2 protein activator; ATP binding activity, peptidyl-prolyl cis-trans isomerase activity, and protein homodimerization activity
RASA1, Cardio and Neuro (3 miRNAs)	mmu-miR-132, mmu-miR-145, mmu-miR-30a	Moderate (67%)	GAP1 family member of GTPase-activating proteins; development and immunity
WNT2B, Cardio and Neuro (3 miRNAs)	mmu-miR-130b, mmu-miR-16, mmu-miR-22	Moderate (67%)	Wingless-type MMTV integration site family, member 2B; cytokine activity and frizzled binding activity
CCNA2, Cardio (3 miRNAs)	mmu-miR-145, mmu-miR-146a, mmu-miR-24	Moderate (67%)	Cyclin; cell cycle regulation, cyclin-dependent kinase 2 activation
CCNG2, Cardio (3 miRNAs)	mmu-miR-135a, mmu-miR-142-5p, mmu-miR-377	Moderate (67%)	Cyclin G2; cyclin-dependent protein serine/threonine kinase regulator activity
CLCF1, Cardio (3 miRNAs)	mmu-miR-135a, mmu-miR-149, mmu-miR-423-3p	Moderate (67%)	Cardiotrophin-like cytokine factor; cytokine activity, positive regulation of B cell activation, positive regulation of astrocyte differentiation, and positive regulation of tyrosine phosphorylation of STAT protein
IGF1R, Cardio and Neuro (6 miRNAs)	mmu-let-7d, mmu-miR-99b, mmu-miR-133a, mmu-miR-145, mmu-miR-16, mmu-miR-486	Moderate (67%)	Insulin-like growth factor receptor; inflammation, cell growth and survival
DNAJC14, Cardio (3 miRNAs)	mmu-miR-125a-5p, mmu-miR-204, mmu-miR-9	Moderate (67%)	DnaJ heat shock protein family, member C14; dopamine receptor binding activity
DNAJC19, Cardio (3 miRNAs)	mmu-miR-125a-5p, mmu-miR-377, mmu-miR-486	Moderate (67%)	DnaJ heat shock protein family, member C19; ATPase activator activity, regulation of cardiolipin metabolic process
FGF16, Cardio (3 miRNAs)	mmu-miR-99b, mmu-miR-467f, mmu-miR-9	Moderate (67%)	Fibroblast growth factor 16; fibroblast growth factor receptor signaling pathway, positive regulation of endothelial cell chemotaxis to fibroblast growth factor, and positive regulation of macromolecule metabolic process
FGF23, Cardio (3 miRNAs)	mmu-miR-34c, mmu-miR-1187, mmu-miR-28	Moderate (67%)	Fibroblast growth factor 23; phosphate homeostasis and vitamin D metabolism
IL11, Cardio (3 miRNAs)	mmu-miR-124, mmu-miR-132, mmu-miR-204	Moderate (67%)	Interleukin 11; cytokine activity and interleukin-11 receptor binding activity
LTB, Cardio (3 miRNAs)	mmu-miR-146a, mmu-miR-181a, mmu-miR-377	Moderate (67%)	Lymphotoxin B; cytokine activity and tumor necrosis factor receptor binding activity and positive regulation of interleukin-12 production
VEGFA, Cardio and Neuro (6 miRNAs)	mmu-miR-126-3p, mmu-miR-150, mmu-miR-16, mmu-miR-29c, mmu-miR-34c, mmu-miR-1187	Moderate (67%)	Vascular endothelial growth factor; endothelial cell growth, angiogenesis, vasculogenesis
PDE7A, Cardio (3 miRNAs)	mmu-miR-125a-5p, mmu-miR-23b, mmu-miR-30a	Moderate (67%)	Cyclic nucleotide phosphodiesterase family member; cAMP hydrolysis and signal transduction
RCAN1, Cardio (3 miRNAs)	mmu-miR-103, mmu-miR-150, mmu-miR-34c	Moderate (67%)	Regulator of calcineurin 1; Ca2+-dependent protein serine/threonine phosphatase regulator activity and identical protein binding activity
UBE2G1, Cardio (3 miRNAs)	mmu-miR-125a-5p, mmu-miR-146a, mmu-miR-26b	Moderate (67%)	Ubiquitin-conjugating enzyme E2G 1; ubiquitin conjugating enzyme activity and ubiquitin ligase binding activity
UBE2V1, Cardio (3 miRNAs)	mmu-miR-101b, mmu-miR-16, mmu-miR-27a	Moderate (67%)	Ubiquitin-conjugating enzyme E2 family member; cell cycle regulation and differentiation
BIRC7, Neuro (3 miRNAs)	mmu-miR-146a, mmu-miR-26b, mmu-miR-377	Moderate (67%)	Baculoviral IAP repeat-containing 7; cysteine-type endopeptidase inhibitor activity involved in apoptotic process and ubiquitin protein ligase activity
BTC, Neuro (3 miRNAs)	mmu-miR-126-5p, mmu-miR-132, mmu-miR-149	Moderate (67%)	Betacellulin, epidermal growth factor family member; differentiation of pancreatic beta cells and plays a protective role in acute pancreatitis
CD80, Neuro (3 miRNAs)	mmu-miR-103, mmu-miR-16, mmu-miR-22	Moderate (67%)	Membrane receptor activated by CD28 or CTLA-4; T-cell proliferation and cytokine production
CXCL10, Neuro (3 miRNAs)	mmu-miR-101b, mmu-miR-135a, mmu-miR-34c	Moderate (67%)	Chemokine (C-X-C motif) ligand 10; chemoattractant activity, chemokine receptor binding activity, and heparin binding activity
GCH1, Neuro (3 miRNAs)	mmu-miR-133a, mmu-miR-34c, mmu-miR-433	Moderate (67%)	GTP cyclohydrolase 1; GTP cyclohydrolase 1 activity, and positive regulation of heart rate
GPR101, Neuro (3 miRNAs)	mmu-miR-124, mmu-miR-34c, mmu-miR-377	Moderate (67%)	G-protein coupled receptor 101; G protein-coupled receptor activity, adenylate cyclase-activating adrenergic receptor signaling pathway
HCRTR2, Neuro (3 miRNAs)	mmu-miR-202-5p, mmu-miR-433, mmu-miR-451	Moderate (67%)	Hypocretin (orexin) receptor 2; orexin receptor activity
HOMER1, Neuro (3 miRNAs)	mmu-miR-145, mmu-miR-152, mmu-miR-22	Moderate (67%)	Homer scaffolding protein 1; scaffold protein binding activity, transmembrane transporter binding activity, and type 5 metabotropic glutamate receptor binding activity, regulation of ion transport and regulation of postsynaptic neurotransmitter receptor activity
NAPG, Neuro (3 miRNAs)	mmu-miR-16, mmu-miR-204, mmu-miR-30a	Moderate (67%)	Soluble NSF attachment gamma; vesicular transport among endoplasmic reticulum and Golgi
NFE2L2, Neuro (3 miRNAs)	mmu-miR-101b, mmu-miR-132, mmu-miR-28	Moderate (67%)	Basic leucine zipper protein family member; antioxidant defense and cytoprotection
PER2, Neuro (3 miRNAs)	mmu-miR-133a, mmu-miR-24, mmu-miR-34c	Moderate (67%)	Period circadian regulator 2; circadian rhythms for metabolism and behavior
PSMB8, Neuro (3 miRNAs)	mmu-miR-125a-5p, mmu-miR-24, mmu-miR-451	Moderate (67%)	Proteasome subunit, beta type 8; endopeptidase activity, fat cell differentiation
RNF41, Neuro (3 miRNAs)	mmu-miR-24, mmu-miR-466 g, mmu-miR-467f	Moderate (67%)	Ring finger protein 41; erythropoietin receptor binding activity and interleukin-3 receptor binding activity
SCN2A, Neuro (3 miRNAs)	mmu-miR-16, mmu-miR-204, mmu-miR-30a	Moderate (67%)	Sodium channel, voltage-gated, type II, alpha; generation and propagation of action potentials in neurons and muscles
SYT4, Neuro (3 miRNAs)	mmu-miR-16, mmu-miR-23b, mmu-miR-30a	Moderate (67%)	Synaptotagmin; dendrite formation (no Ca2+ binding)
TAF9B, Neuro (3 miRNAs)	mmu-let-7d, mmu-miR-125a-5p, mmu-miR-202-5p	Moderate (67%)	TATA-box binding protein associated factor 9B; protein heterodimerization activity, RNA polymerase II general transcription initiation factor activity, negative regulation of transcription by RNA polymerase II, positive regulation of cell growth, and protein stabilization
RAB9B, Neuro (6 miRNAs)	mmu-miR-103, mmu-miR-152, mmu-miR-16, mmu-miR-202-5p, mmu-miR-1187, mmu-miR-367	Borderline (50%)	RAB9B, member RAS oncogene family; protein binding activity, retrograde transport, endosome to Golgi
RGS8, Neuro (6 miRNAs)	mmu-miR-103, mmu-miR-16, mmu-miR-1929, mmu-miR-23b, mmu-miR-27a, mmu-miR-34c	Borderline (50%)	Regulator of G-protein signaling 8; G-protein alpha subunit binding and activator activity, G- protein coupled acetylcholine receptor signaling pathway, positive regulation of GTPase activity, and regulation of dopamine receptor signaling pathway
BCL2, Cardio and Neuro (4 miRNAs)	mmu-miR-143, mmu-miR-16, mmu-miR-181a, mmu-miR-34c	Borderline (50%)	B-cell lymphoma 2 apoptosis regulator; programmed cell death/apoptosis
IL36G, Cardio and Neuro (4 miRNAs)	mmu-miR-143, mmu-miR-146a, mmu-miR-29c, mmu-miR-1187	Borderline (50%)	Interleukin 36G; cytokine activity
IL6, Cardio and Neuro (4 miRNAs)	mmu-miR-149, mmu-miR-191, mmu-miR-365, mmu-miR-574-3p	Borderline (50%)	Cytokine; cell growth and differentiation, inflammation, hematopoiesis
MAPK7, Cardio and Neuro (4 miRNAs)	mmu-miR-143, mmu-miR-145, mmu-miR-200c, mmu-miR-24	Borderline (50%)	Mitogen-activated protein kinase; cell signal integration and downstream signaling
MET, Cardio and Neuro (4 miRNAs)	mmu-miR-130b, mmu-miR-23b, mmu-miR-30a, mmu-miR-34c	Borderline (50%)	Hepatocyte growth factor receptor/receptor tyrosine kinase; cell development and survival
MYC, Cardio and Neuro (4 miRNAs)	mmu-let-7d, mmu-miR-145, mmu-miR-24, mmu-miR-34c	Borderline (50%)	Nuclear phosphoprotein; cell cycle progression and apoptosis
NPR3, Cardio and Neuro (4 miRNAs)	mmu-miR-103, mmu-miR-150, mmu-miR-16, mmu-miR-30a	Borderline (50%)	Natriuretic peptide receptor 3; diuresis and blood pressure regulation
P2RY1, Cardio and Neuro (4 miRNAs)	mmu-miR-190, mmu-miR-34b-3p, mmu-miR-365, mmu-miR-467f	Borderline (50%)	Purinergic receptor P2Y, G-protein coupled 1; A1 adenosine receptor binding activity, G-protein-coupled purinergic nucleotide receptor activity, and adenyl ribonucleotide binding activity
PDE1A, Cardio and Neuro (4 miRNAs)	mmu-miR-135a, mmu-miR-22, mmu-miR-24, mmu-miR-377	Borderline (50%)	Phosphodiesterase 1A, calmodulin-dependent; Ca2+ and calmodulin regulated 3’,5’-cyclic-GMP phosphodiesterase activity, and signal transduction
TP53, Cardio and Neuro (4 miRNAs)	mmu-let-7d, mmu-miR-125a-5p, mmu-miR-30a, mmu-miR-34c	Borderline (50%)	Tumor suppressor protein; cell cycle arrest, apoptosis, and senescence
APLN, Cardio (4 miRNAs)	mmu-miR-103, mmu-miR-124, mmu-miR-125a-5p, mmu-miR-16	Borderline (50%)	Adipokine; adipocyte secretion and cardiovascular development/function
FGF7, Cardio (4 miRNAs)	mmu-miR-143, mmu-miR-16, mmu-miR-34c, mmu-miR-486	Borderline (50%)	Fibroblast growth factor; cell proliferation, differentiation, and survival
SNTB2, Cardio (4 miRNAs)	mmu-miR-124, mmu-miR-135a, mmu-miR-16, mmu-miR-34c	Borderline (50%)	Syntrophin β2/dystrophin; membrane protein and cytoskeleton localization
UBE2D1, Cardio (4 miRNAs)	mmu-miR-101b, mmu-miR-142-5p, mmu-miR-152, mmu-miR-27a	Borderline (50%)	Ubiquitin conjugating enzyme E2 D1; degradation of short-lived and abnormal proteins
CPLX2, Neuro (4 miRNAs)	mmu-miR-125b-3p, mmu-miR-135a, mmu-miR-34c, mmu-miR-1187	Borderline (50%)	Complexin 2; Ca2+-dependent protein binding activity and syntaxin-1 binding activity, positive regulation of synaptic activity and regulation of synaptic vesicle fusion to presynaptic active zone membrane
CYCS, Neuro (4 miRNAs)	mmu-miR-145, mmu-miR-29c, mmu-miR-433, mmu-miR-1187	Borderline (50%)	Cytochrome c, somatic; heme binding activity
NTRK3, Neuro (4 miRNAs)	mmu-miR-99b, mmu-miR-128, mmu-miR-34c, mmu-miR-9	Borderline (50%)	Neurotrophic tyrosine receptor kinase family member; development and cell survival/differentiation
POLR2K, Neuro (4 miRNAs)	mmu-miR-126-5p, mmu-miR-204, mmu-miR-2183, mmu-miR-434-3p	Borderline (50%)	Polymerase (RNA) II (DNA directed) polypeptide K; DNA binding activity, DNA-directed 5’-3’ RNA polymerase activity and zinc ion binding activity
S1PR3, Neuro (4 miRNAs)	mmu-miR-125a-5p, mmu-miR-151-5p, mmu-miR-34c, mmu-miR-486	Borderline (50%)	Sphingosine-1-phosphate receptor 3; vascular and heart development, mediates HDL and HDL-associated lysophospholipid-induced vasorelaxation, and coordinates with other lysophospholipid receptors in the process of angiogenesis
SNAP23, Neuro (4 miRNAs)	mmu-let-7d, mmu-miR-124, mmu-miR-34c, mmu-miR-1187	Borderline (50%)	Synaptosomal-associated protein; SNAP receptor activity and syntaxin binding activity
TSC1, Neuro (4 miRNAs)	mmu-miR-126-3p, mmu-miR-130b, mmu-miR-34c, mmu-miR-451	Borderline (50%)	TSC complex subunit 1; ATPase inhibitor activity and protein N-terminus binding activity, associative learning, negative regulation of ATPase activity, and protein stabilization
DVL3, Cardio (4 miRNAs)	mmu-let-7d, mmu-miR-129-3p, mmu-miR-204, mmu-miR-1187	Borderline (50%)	Disheveled segment polarity protein; protein-macromolecule adaptor activity, canonical Wnt signaling pathway via JNK cascade
HIF1AN, Cardio (4 miRNAs)	mmu-let-7d, mmu-miR-125b-3p, mmu-miR-125a-5p, mmu-miR-23b	Borderline (50%)	Hypoxia-inducible factor 1, alpha subunit inhibitor; 2-oxoglutarate-dependent dioxygenase activity, NF-kappaB binding activity, and transition metal ion binding activity.

Cardio and Neuro Strings: Target numbers and extent of overlap before and during AD

Emerging evidence points to AD as a neurovascular disorder [2], whereby the brain parenchyma is dependent on the metabolic nourishment provided by the cerebral microcirculation and vice versa. Based on our view from cerebrovascular samples isolated from 3xTg-AD mice, there is a total of 964 discrete mRNA targets among general Cardio and Neuro signaling pathways to consider for best marking the development of AD pathology. The Cardio string revealed 711 targets, ranging from 1 to 80 in number regulated by each miRNA (mean±SEM: 20.69±2.29) (Fig. 1). In turn, each Cardio target is regulated by a range of 1 to 11 miRNAs (1.78±0.04) (Supplementary Table 1). The Neuro string indicates 1,163 targets, ranging from 5 to 131 per miRNA (32.56±3.35) (Fig. 1). Accordingly, each Neuro target is regulated by a range of 1 to 11 miRNAs (1.71±0.03) (Supplementary Table 2). Overlap among Cardio and Neuro targets ranges from 1 to 56 (13.54±1.52) (Fig. 1) corresponding to a fractional overlap among the Cardio and Neuro marker pools as 0.66±0.02 and 0.41±0.01 respectively (Fig. 2A). The fraction of shared Cardio targets with Neuro was higher (p<0.0001) relative to vice versa (shared Neuro with Cardio) throughout conditions as miRNAs that changed in expression with AD pathology (Cardio: 0.68±0.03; Neuro: 0.41±0.02) and those stable in expression throughout (Cardio: 0.66±0.02; Neuro: 0.40±0.02) (Fig. 2B). In turn, note that conditions of AD pathology do not alter (p>0.90) the fraction of shared targets among pathways for either Cardio or Neuro (Fig. 2B). Altogether, these data support a high level of integration of the cardiovascular system with the nervous system in the blood vessels of the brain that is retained with AD. Our next goal was to determine the identity of the individual molecular markers themselves that are regulated by respective miRNAs and whether the onset of AD could be tracked accordingly.

Let-7d, miR-181a, miR-132, and miR-99a: shared mRNA targets

We found that let-7d, miR-181a, miR-132, and miR-99a are significantly downregulated in aging males, and thereby mark the onset of the Aβ/AD in 3xTg-AD male mice (Table 1) [10]. Out of all the miRNAs that change in expression with AD, let-7d has the highest number of targets for Cardio (=69), Neuro (=89), and respective overlap (=43) (Table 1 and Fig. 1). For Cardio and Neuro, miR-181a has 15 and 24 targets respectively (overlap=7); miR-132 has 12 and 22 targets respectively (overlap=8); and miR-99a marks transition from CI to A [10] and has 10 and 16 targets respectively (overlap=5) (Table 1 and Fig. 1).

For Cardio, the most noteworthy markers are V-Ki-Ras2 Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS); TNF Superfamily Member 10 (TNFSF10) among let-7d and miR-181a; IGF1R among let-7d and miR-99; and TNFRSF11B among miR-181a and miR-132 (Fig. 3A). Examples of common families shared among at least two of these miRNAs include the Ras Homolog Family (RHO) among let-7d and miR-181a; TNFSF among let-7d, miR-132, and miR-181a; DnaJ Heat Shock Protein Family Hsp40 (DNAJ), Fibroblast Growth Factor (FGF) and Frizzled Class Receptor (FZD) among let-7d and miR-99; and the Matrix Metallopeptidase (MMP) family among miR-132 and miR-181a. Further, the Interleukin (IL) family spans across let-7d, miR181a, and miR-132.

For Neuro, the same relationships remain for KRAS and IGF1R with an addition of GRIN2B shared among let-7d and miR-181a and Polymerase RNA II Subunit D (POLR2D) among let-7d and miR-132 (Fig. 3B). miR-132 and let-7d also share members of the SLC family. Let-7d and miR-181a share the “a disintegrin and metalloproteinase with thrombospondin motif” (ADAMTS) family (Fig. 3B). Further, the G Protein-Coupled Receptor family (GPR) spans across let-7d, miR-132, and miR-99.

miR-151-5p: interactions with let-7d and miR-99 for Cardio and let-7d and miR-181a for Neuro

While not discerning biological sex differences, miR-151-5p is also a strong indicator for overall AD while marking the onset of the Aβ with sexes combined (Table 1) [10]. miR-151-5p has 11 and 32 targets for Cardio and Neuro respectively (overlap=8) (Table 1 and Fig. 1). For Cardio, miR-151-5p shares the Protein Phosphatase (PPP) family with let-7d and miR-99 (Fig. 4A). Also, miR-151-5p targets the muscarinic cholinergic receptor target CHRM2 while let-7d regulates the nicotinic cholinergic receptor target CHRNA7. For Neuro, it is worth noting that miR-151-5p also targets a member of the GRIN family as GRIN1 with noting that let-7d and miR-181a Share GRIN2B as well (Fig.4B). As with let-7d, miR-132, and miR-99, miR-151-5p also shares a GPR family member as GPR173 (Fig. 4B).

Fig. 4.

Overlap of mRNA targets among mmu-miR-151-5p with other similar miRNAs for AD pathology for Cardio and Neuro. Bold text: precise targets that overlap among respective miRNAs; Bold, underlined text: precise targets that overlap among respective miRNAs while similar isoforms among the same gene family with at least one other miRNA; italicized text: similar isoforms among the same gene family; italicized, underlined text: similar isoforms among the same gene family for all miRNAs illustrated. A)Cardio: Note precise overlap for IGF1R. Shared families among at least two miRNAs include CACN, DNAJ, FGF, FZD, IGF, IL, MAP3K, MAPK, and PPP. B) Neuro: Note precise overlap for GRIN2B and KRAS. Shared families among at least two miRNAs include ADAMTS, BCL2, CACN, GABR, GPR, GRIN, KCNJ, MAPK, MAP3K, PPP, PRK, PTG, RAS, and RHO. See Table 1 for all details regarding each miRNA and original data are reported in [10].

miR-23, miR-126-3p, and miR-150: shared mRNA targets

miR-23a/b and miR-126-3p mark overall AD versus Pre-AD through significant downregulation in males only, whereas miR-150 indicates AD for both males and females (Table 1) [10]. For Cardio and Neuro, miR-23 has 20 and 33 targets respectively (overlap=14); miR-126-3p has 9 and 16 targets respectively (overlap=8); and miR-150 has 13 and 17 targets respectively (overlap=10) (Table 1 and Fig. 1). For both Cardio and Neuro, VEGFA and Myosin Heavy Chain 1 (MYH1) emerge as shared among miR-126-3p and miR-150 and miR-23 and miR150 respectively (Fig. 5). miR-23 and miR-126-3p share the Regulator of G-protein Signaling (RGS) and SYT families along the Neuro string (Fig. 5B). It is also worth noting that miR-150 targets GRIN2B and PDGFB as common with let-7d and miR-181a and let-7d only, respectively.

Fig. 5.

Overlap of mRNA targets among mmu-miR-23, mmumiR-126-3p, and mmu-miR-150 for AD pathology. Bold text: precise targets that overlap among respective miRNAs; italicized text: similar isoforms among the same gene family. A)Cardio:Note precise overlap for MYH1 and VEGFA. Shared families among at least two miRNAs include IL, MYH, and VEGF. B) Neuro: Note precise overlap for MYH1 and VEGFA. Shared families among at least two miRNAs include MYH, RGS, SYT, and VEGF. See Table 1 for all details regarding each miRNA and original data are reported in [10].

miR-27a and miR-135a: shared targets and interactions with miR-23 for Cardio and Neuro

Although relatively weak indicators of subtle AD pathology progression, miR-27a and miR-135a are decreased in expression in AD versus Pre-AD animals (Table 1) [10]. For Cardio and Neuro, miR-27a has 43 and 62 targets respectively (overlap=29); and miR-135a has 27 and 39 targets respectively (overlap=17) (Table 1 and Fig. 1). For both Cardio and Neuro, the SMAD family is shared among miR-23, miR-27a, and miR-135a with SMAD5 as the most notable (Fig. 6). Forkhead Box O1 (FOXO1) and the Myocyte Enhancer Factor (MEF) family (MEF2A and MEF2C) are indicated among miR-27a and miR-135a as well (Fig. 6). Note that miR-126-3p targets a MEF family member (MEF2B) as well (Table 1). The PDE family is shared among miR-27a and miR-135a along with miR-23 (Fig. 6). As unique to Cardio, PKIA is a common target among miR-23 and miR-27 (Fig. 6A). Neuro continues to illustrate the importance of the SLC and GRIN families during AD pathology as shared by miR-27a and miR-135a (Fig. 6B). With a match among miR-27a and miR-135a as SYT3, other AD-marking miRNAs as miR-23, miR-126-3p, and miR-151-5p, miR-27a and miR-135a also share members of the SYT family (Table 1).

Fig. 6.

Overlap of mRNA targets among mmu-miR-23, mmumiR-27a, and mmu-miR-135 for AD pathology. Bold text: precise targets that overlap among respective miRNAs; Bold, underlined text: precise targets that overlap among respective miRNAs while similar isoforms among the same gene family with at least one other miRNA; italicized text: similar isoforms among the same gene family; italicized, underlined text: similar isoforms among the same gene family for all miRNAs illustrated. A) Cardio: Note precise overlap for FOXO1, MEF2C, PKIA, and SMAD3/4/5. Shared gene families among at least two miRNAs include CACN, DNAJ, FGF, MEF, MYL, PDE, and SMAD. B) Neuro: Note precise overlap for FOXO1, MEF2C, NOTCH1, RGS8, SMAD3/4/5, SNAP25, and SYT3. Shared gene families among at least two miRNAs include CACN, CXCL, FOXO, GPR, GRIN, MEF, MYL, NOTCH, NRP, PDE, RGS, SLC, SMAD, SNAP, and SYT. See Table 1 for all details regarding each miRNA and original data are reported in [10].

miR-133a: interactions with let-7d and miR-99 (Cardio) and let-7d, miR-99, and miR-132 (Neuro)

miR-133a uniquely marks onset of CI in female animals but has limited utility for indicating overall AD pathology (Table 1) [10]. miR-133a has 25 and 45 targets for Cardio and Neuro respectively (overlap=16) (Table 1 and Fig. 1). For Cardio and Neuro, it is notable that miR-133a shares IGF1R with let-7d and miR-99 along with members of the PPP family (Fig. 7). Further, miR-133a shares the PDE family with let-7d, miR-23, miR-27a, and miR-135a (Table 1). A Neuro marker as Pregnancy-Associated Plasma Protein A (PAPPA) is shared among miR-133a and let-7d (Fig. 7B). In addition, Neuro continues to illustrate the importance of the SLC family for AD pathology among miR-133a, miR-132, and let-7d (Fig. 7B). Further, the SYT family is highlighted as targets of miR-133a as common with miR-23, miR-27a, miR-126-3p, and miR-151-5p (Table 1). Other Neuro markers include Notch Receptor 1 (NOTCH1), RGS8, and Synaptosome Associated Protein 25 (SNAP25) as shared among miR-23 and miR-27a (Fig. 7B).

Fig. 7.

Overlap of mRNA targets among mmu-miR-133a with other similar miRNAs for AD pathology for Cardio and Neuro. Bold text: precise targets that overlap among respective miRNAs; italicized text: similar isoforms among the same gene family; italicized, underlined text: similar isoforms among the same gene family for all miRNAs illustrated. A) Cardio: Note precise overlap for IGF1R. Shared gene families among at least two miRNAs include CDC, DNAJ, FGF, FZD, IGF MAP3K, PDE, PPP, RAS, and RHO. B) Neuro: Note precise overlap for IGF1R, PAPPA, POLR2D, and SLC6A1. Shared gene families among at least two miRNAs include ADGR, FGF, FZD, GPR, IGF, IL, POLR, PPP, RAS, RHO, SCN, SLC, and TGFB. See Table 1 for all details regarding each miRNA and original data are reported in [10].

Other notable miRNAs for AD pathology: miR-690, miR-29c, miR-34b-3p, miR-129-3p, and miR-539

Other miRNAs that indicate AD pathology include miR-690, miR-29c, miR-34b-3p, miR-129-3p, and miR-539 (Table 1) [10]. miR-690 has 5 and 7 targets for Cardio and Neuro respectively (overlap=4) (Table 1). For both Cardio and Neuro, miR-690 shares Ras-related Protein R-RAS 2 (RRAS2) with miR-23 and the Caspase (CASP) family with let-7d, miR-135a, miR-29c, and miR-129-3p. For Neuro only, Intersectin 1 (ITSN1) is shared among miR-690 and miR-181a. miR-29c has 32 and 65 targets for Cardio and Neuro respectively (overlap=24) (Table 1). For both Cardio and Neuro, miR-29c contains prominent markers for AD pathology such as PDGFA and PDGFB. Also, the Cell Division Cycle 42 (CDC42) marker is shared among miR-29c and miR-133a. miR-34b-3p has 4 and 8 targets for Cardio and Neuro respectively (overlap=3), whereas miR-539 has 3 and 6 targets respectively (overlap=2) (Table 1). For Neuro, the Gamma-Aminobutyric Acid Type A Receptor Subunit α 4 (GABRA4) marker is shared exclusively among miR-34b-3p and miR-539. Also, both miRNAs have a transmembrane receptor or ion channel target as Purinergic Receptor P2Y1 (P2RY1) and Potassium Inwardly Rectifying Channel Subfamily J member 6 (KCNJ6) respectively. miR-129-3p has 19 and 23 targets for Cardio and Neuro respectively (overlap=11) (Table 1). A noteworthy observation is that miR-129-3p also targets GRIN2B with other AD miRNA pathology markers such as let-7d, miR-135, miR-150, and miR-181a. In addition, miR-129-3p targets the SMAD family as with miR-23, miR-27a, miR-132, and miR-135. miR-129-3p shares KCNJ1 with miR-126-3p and the KCNJ family in general with others as let-7d, miR-135a, miR-151-5p, and miR-539.

miRNAs and respective mRNAs that indicate role of biological sex during Pre-AD or AD

A total of 29 miRNAs distinguish between males or females during Pre-AD and/or AD, whereby 22 do not mark progression of AD pathology itself per se (Table 1) [10]. One of such miRNAs includes the smooth muscle/pericyte-specific miR-145 [12, 18]. Of note, all of the 5 miRNAs that selectively indicate differences in the Young group (miR-99b, miR-101b, miR-133a, miR-204, and miR-377) are higher (p<0.05) in expression in females versus males. For Pre-AD overall, 19 miRNAs (miR-23b, miR-34b-3p, miR-34c, miR-99b, miR-142-5p, miR-145, miR-149, miR-150, miR-200b/c, miR-202-5p, miR-377, miR-423-3p, miR-466g, miR-467f, miR-539, miR-574-3p, miR-1187, miR-1942, and miR-2183) have a higher (p<0.05) expression in females versus males, whereas only 5 (miR-16, miR-22, miR-132, miR-382, miR-451) are lower (p<0.05) in females versus males. Only 2 miRNAs were different in expression among males and females during AD only as miR-125a-5p (females>males) and miR-146a (females<males).

Overall, the most prominent targets for marking biological sex itself include HDAC8, HSPA1A/1B, IL6R, Potassium Calcium-Activated Channel Subfamily M Regulatory β Subunit 1 (KCNMB1), PDGFD, and Rac Family Small GTPase 1 (RAC1) for both Cardio and Neuro; Activating Transcription Factor 6 (ATF6) for Cardio only; Arrestin β1 (ARRB1), Cholecystokinin (CCK), CFL2, Colony Stimulating Factor 1 Receptor (CSF1R), Erb-B2 Receptor Tyrosine Kinase 3 (ERBB3), ORAI2, Retinoic Acid Receptor-Related Orphan Receptor α (RORA), SYT1/2, SCN2B, SIRT1, and Vesicle Transport Through Interaction With T-SNAREs 1B (VTI1B) for Neuro only (Table 4). Note that three members each of the SYT (SYT1/2/4) and SCN (SCN2A/2B/8A) families mark differences among males and females.

Strong, moderate, and borderline mRNA markers for AD pathology and biological sex

The most highly targeted markers across both Cardio and Neuro include the Phosphatase and Tensin Homolog (PTEN, 11 miRNAs for each) and Transforming Growth Factor β Receptor 2 (TGFBR2, 8 miRNAs), whereby both markers are weak indicators of AD pathology (≤27%) and biological sex differences (≤36%) (Table 2). PDGFB is strong (75%, 4 regulating miRNAs) and SMAD3 is moderate (60%, 5 regulating miRNAs) for AD (Table 3). Other moderate markers for AD throughout both Cardio and Neuro while regulated by 3 miRNAs include CDC42, FGF1, G Protein Subunit Gamma 5 (GNG5), MEF2C, MYH1, PDGFA, and Phospholipase A2 Group IIF (PLA2G2F) (Table 3). “Borderline” AD markers (≈50%) regulated by 4 or more miRNAs include FOXO1, IGF1R, SMAD4/5, Sp1 transcription factor (SP1) and VEGFA (Tables 2 and 3). Note that CDC42, GNG5, IGF1R, MYH1, and VEGFA are all moderate (67%) markers for biological sex as well (Table 4).

For Cardio-specific, PKIA and Ubiquitin Conjugating Enzyme E2 D2 (UBE2D2) are regulated by the most miRNAs (=7) while weak markers for AD pathology overall (43% and 14% respectively) and weak to moderate for biological sex (57% and 43% respectively) (Table 2). TNFRSF11B is strong (75%) for AD and is regulated by 4 miRNAs (Table 3). Moderate Cardio AD markers regulated by 3 miRNAs include the Collagen Type Iα Chain members (COL1A1/A2), an E3 Ubiquitin Protein Ligase (NEDD4), PDE8B, and Regulator of Calcineurin 2 (RCAN2) (Table 3). Borderline AD markers regulated by 4 miRNAs for both AD and biological sex include Dishevelled Segment Polarity Protein 3 (DVL3) and Hypoxia Inducible Factor 1 Subunit α Inhibitor (HIF1AN) (Tables 3 and 4).

For Neuro-specific, GRIN2B and RGS17 are regulated by the most miRNAs (=9), whereby GRIN2B is a moderate (56%) marker for AD pathology in contrast to RGS17 (11%) (Tables 2 and 3) Both GRIN2B and RGS17 are weak markers for biological sex as 44% and 33% respectively. SYT3 is regulated by 4 miRNAs while strong (75%) for AD pathology (Table 3). Moderate markers of AD pathology regulated by 5 miRNAs include CFL2 and SLC6A1. Other moderate markers regulated by 3 miRNAs include ADAMTS14/15, GPR55, KCNJ1, PAPPA, POLR2D, RGS1, and Vesicle Associated Membrane Protein 2 (VAMP2). Borderline markers include ADAMTS2, β-Secretase 1 (BACE1), Ectonucleoside Triphosphate Diphosphohydrolase 7 (ENTPD7), SCN2B, and Versican (VCAN) (Table 3). The SCN (SCN2A/2B/8A) and, except for SYT3, SYT (SYT1/2/4) families generally indicate differences in biological sex (60%–100%) (Table 4). Other sex markers include CFL2 as strong (100%) and SLC6A1, PAPPA, POLR2D, and VAMP2 as moderate (Table 4).

Other strong (100%) markers for AD while regulated by only 2 miRNAs include Activin A Receptor Type 1 (ACVR1C), Phosphoinositide-3-Kinase Regulatory Subunit 2 (PIK3R2), RRAS2, and SMAD9 for Cardio and Neuro; Diaphanous Related Formin 2 (DIAPH2), DNAJB7, and TNFSF10 for Cardio; and ADAMTS12, Fas Cell Surface Death Receptor (FAS), FKBP Prolyl Isomerase 1A (FKBP1A), FosB Proto-Oncogene AP-1 Transcription Factor Subunit (FOSB), GABRA4, Glial Cell Derived Neurotrophic Factor (GDNF), GPR82, ITSN1, Lipase H (LIPH), RGS16, and Stathmin 2 (STMN2) for Neuro.

Markers absent for indicative value among AD pathology and biological sex

The most highly targeted marker that is absent (0%) for AD pathology is the Ras Family Small GTP Binding Protein (RAP1B, 7 miRNAs) (Table 2). Others in this category while regulated by 6 miRNAs include UBE2W (Cardio), H3.3 Histone A/B (H3–3A/B), Ras-Related Protein Rab-9B (RAB9B), and RGS4 (Neuro). All others regulated by at least 4 miRNAs include IL6, GNG10, and Mitogen-Activated Protein Kinase 7 and 14 (MAPK7/14) (Cardio and Neuro); Apelin (APLN), and FGF7 (Cardio); and ERBB3, KCNJ2, and POLR2K (Neuro).

For biological sex, absent markers regulated by 4 miRNAs include the apoptosis regulator BCL2-Associated X Protein (BAX), CASP3, FOXO1, and Ribosomal Protein S6 Kinase A5 (RPS6KA5) for Cardio and Neuro; Cyclin Dependent Kinase Inhibitor 1A (CDKN1A) for Cardio; and BACE1, Sphingosine-1-Phosphate Receptor 1 (S1PR1), and VCAN for Neuro. Markers absent for both AD pathology and biological sex are regulated by 4 miRNAs at most and include ACVR1, Bone Morphogenetic Protein 5 (BMP5), and Ras Related Dexamethasone Induced 1 (RASD1) for Cardio and Neuro; IL10RB for Cardio; and 5-Hydroxytryptamine Receptor 1F (HTR1F), Neuronal Differentiation 1 (NEUROD1), and Pro-Apoptotic WT1 Regulator (PAWR) for Neuro.

DISCUSSION

The development of dementia has eluded science and medicine for well over 150 years [32], with AD proper identified by 1910 with presence of visible plaques and/or neurofibrillary tangles in brain histology specimens [33]. It was not until 1980 that at least 100 peer-reviewed studies/year were dedicated to understanding AD and, since then, that number has increased exponentially to currently over 16,000/year for 2022. Recently, focus has shifted for the etiology of AD from primarily a neurological disease to one that involves disruption in healthy neurovascular interactions [2, 26]. There remains a clear need in locating and identifying molecular signatures of AD development from original genetic material as DNA and throughout to post-translational modifications of functional proteins. Our previous work focused on a comprehensive analysis and discussion of cerebrovascular miRNAs in particular that are consistently expressed throughout adulthood in male and female 3xTg-AD mice [10]. miRNAs in general are promising biomarkers due to their novelty in modern molecular biology research while stable in the blood circulation [9]. We found that a select set of cerebrovascular miRNAs were informative for tracking early development of AD pathology [10]. Thus, the current study was an in-depth analysis of the known cardiovascular and nervous system mRNA targets regulated by the cerebrovascular miRNAs that mark onset of AD versus miRNAs that remained stable in expression throughout. In addition, we examined molecular targets that differed among males and females during Pre-AD or AD conditions. There was a significant overlap (≥40%) among Cardio and Neuro pathways and mRNA targets regulated by cerebrovascular miRNAs detected in the brains of 3xTg-AD mice, supporting neurovascular coupling at the molecular level. Prominent mRNA markers indicating AD pathology altogether support excitotoxicity (GRIN2B, SLC6A1, SYT3) coupled with vascular remodeling (e.g., FGF1, PDE8B, PDGFA/B, SMAD3/4/5, VEGFA) and inflammation (TNFRSF11B). Further, differences among males and females center on cardiac function (e.g., SCN2B), neuronal growth and neurotransmission (e.g., NAPG, NTRK3, SCN2A/8A SLC6A1, SYT1/2/4, VTI1B), transcriptional regulation and protection of the proteome (e.g., HDAC8, HSPA1B, SIRT1), and cell cycle/proliferation (e.g., CCNA2, CCND1, ERBB3, FGF16, IGF1R, UBE2G1/V1, VEGFA). Fundamental and translational implications of these findings in the context of AD pathology are further discussed in detail below.

Abundant overlap of targets among the cardiovascular and nervous system pathways: support for neurovascular integration at the molecular level

Regardless of AD pathology, most (≈70%) of the cardiovascular molecular target pool overlaps with that of the nervous system, whereas the vice versa for the nervous system with cardiovascular system is significantly less (≈40%) (Figs. 1 and 2). These data support an enhanced integration of cardiovascular signaling targets with that of the nervous system during conditions of health and disease. As a whole, such overlap is consistent with a revised view that both vascular and neurodegenerative factors are integral to the development of dementia [34, 35].

Prominent miRNAs for marking AD pathology and major common targets

The miRNAs that marked AD pathology were organized into tiers based on points of significance among individual phases of pathological development (Young versus CI versus Aβ versus AβT), overall AD (Aβ and AβT) versus Pre-AD (Young and CI) in accord with biological sex. We sought common targets among each tier as well among all miRNAs that alter in expression with AD versus the others that remain stable. Let-7d, miR-181a, miR-132, miR-99, and miR-151-5p scored the highest with highlights for GRIN2B, IGF1R, KRAS, POLR2D, TNFSF10, and TNFRSF11B (Figs. 3 and 4). Next, the addition of miR-126-3p and miR-150 revealed MYH1 and VEGFA (Fig. 5). miR-23 combined with miR-27a and miR-135a pointed to the SMAD (SMAD3/4/5/9) and PDE (PDE1A/4B/6D/7A/7B/8B) families (Fig. 6) with an emphasis on SMAD3 and PDE8B in particular for AD pathology (Table 3). miR-133a added SLC6A1 with analysis of miR-132 and further illustration of the importance of IGF1R when combined with let-7d and miR-99 (Fig. 7). As the most significant miRNA, let-7d regulates 44% (12 out of 27) of the mRNA targets shared among≥3 miRNAs that best indicate AD pathology (Table 3). Overall, in our hands for 3xTg-AD animals, let-7d is arguably the strongest miRNA marker for AD pathology. This observation is consistent with findings for human subjects [36, 37].

Potential role for cellular excitotoxicity: GRIN family, SLC6A1, and SYT3

GRIN2B is the most prominent marker for the highest number of regulating miRNAs (=9) that coincide with onset of AD pathology (56%). As a whole, 50% (7 out of 14) of the GRIN family members (GRIN1/2A/2B/2C/2D) correspond to miRNAs that alter in expression with AD pathology. The significance of the GRIN family is that they code for NMDA receptor subunits as integral to aberrant neuronal excitation/synaptic plasticity (GRIN2B) [38] and a more recently recognized role for impaired cerebral blood flow regulation (GRIN1) [39] regulated by miR-151-5p during AD pathology. Heightened expression for GRIN2B and SLC6A1 can also coexist during conditions of MCI and AD [40]. Their combined role supports neuroexcitotoxicity as SLC6A1 terminates GABA activity (hyperpolarization/repolarization), thus removing a major brake to membrane excitation (depolarization) while exacerbating the activity of excitatory Ca²+-permeant NMDA receptors sensitive to glutamate [41]. Further, in tandem, SYT3 (Table 3) may also contribute to enhanced high frequency presynaptic exocytosis of glutamate as well [42].

Potential contribution of IGF/IGF1R signaling to AD pathology

Although a borderline indicator of AD pathology progression overall in our hands (Table 3), IGF/IGF1R signaling remains noteworthy as regulated by let-7d, miR-27a, miR-99, and miR-133a. Insulin resistance and dysregulation of IGF/IGF1R signaling have been demonstrated in postmortem brain tissues of humans with a prior diagnosis of AD [43]. Further, small molecule inhibition of IGF1R in the APP/PS1 mouse model (human mutant amyloid precursor protein + presenilin−1) using picropodophyllin attenuates development of AD [44]. With>40 years of studies examining a link between Type II Diabetes (T2D) and AD [45, 46] and a recent sharp increase of T2D in the general population [47], the role of IGF/IGF1R will likely remain as a pertinent target for treatment of dementia for the foreseeable future [48].

Angiogenesis gone awry: PDGF, PDE, SMAD, and VEGF families

As integral to angiogenic signaling, our data point to consistent roles of the PDGF, PDE, SMAD, and VEGF families during development of AD pathology. PDGFB is crucial for pericyte development [49] and PDGFA of the glial vascular unit in particular is associated with late stage AD neuropathology [50]. PDE8B is upregulated in AD brains of human subjects [51]. Further, APP C-terminal fragments can induce a specific increase in PDE8B in vitro in Chinese Hamster Ovary (CHO) cells [52]. Also, pharmacological inhibition of SMAD3 enhances amyloid clearance from the brains of APP/PS1 mice [53]. Pathological VEGF signaling has also been identified with AD and, in particular, VEGFA can stimulate abnormal angiogenesis in the brain, resulting in capillary stalling and impaired cerebral blood flow [24, 54]. It is worth noting that VEGFA is central to cancer pathology as well, whereby poor-quality vessels are produced with features of irregular, tortuous branching and hyperpermeability [55]. Altogether, cumulative molecular and integrative evidence suggests that enhanced angiogenic signaling is not necessarily favorable for maintaining healthy perfusion of the brain during development of AD and advanced amyloidosis.

Tumor necrosis factor as a therapeutic target

Tumor necrosis family members also play a role in AD pathology in ours tudies (Table 3). As a key example, TNFSF10 is a proapoptotic cytokine regulated by strong miRNA indicators of AD pathology as let7d and miR-181a (Table 1). Indeed, neutralization of TNFSF10 in 3xTg-AD mice using an antibody from 3 to 9 months of age improves cognition while reducing hippocampal amyloid burden and inflammation [56]. As regulated by miR-132, miR-135a, and miR181a, TNFRSF11B (or osteoprotegerin) was a strong indicator of AD pathology in our studies (Table 3). TNFRSF11B has also been identified as a potential therapeutic target for MCI and AD in human subjects with crossover applications as cardiovascular disease and osteoporosis [57, 58].

Endothelin signaling and AD pathology

Let-7d regulates Endothelin 1 (EDN1) as a potent vasoconstrictor integral to AD pathology [59, 60]. According to our current data set, endothelin receptor signaling during AD pathology can be delineated by examining the roles of miR-135 [Endothelin Receptor Type A (EDNRA)] and miR-29c (EDNRB). Also, the reader should also bear in mind that EDNRA is also regulated by the stably expressed miRNAs as miR-30a and miR-128 (Table 1).

Role of vascular cell type: Endothelial and smooth muscle/pericyte markers

Levels of “endothelial-specific” miRNAs such as miR-23a/b [12], miR-27a [13, 14], and miR-126-3p [15, 16] are significantly decreased in AD relative to pre-AD animals. miR-27a in particular targets 8 of the strongest AD markers as FGF1, MEF2C, NEDD4, RCAN2, RGS1, SLC6A1, SMAD3, and SYT3 (Table 3). These markers generally encompass vascular growth and contractility. The smooth muscle/pericyte-specific miRNAs such as miR-143 [17] and miR-145 [12, 18] remain stable throughout all animal groups (Table 1) [10]. With the exception of GRIN2B (regulated by miR-143) and IGF1R (regulated by miR-145), neither miRNA regulates mRNA markers of AD (Table 3). As consistent with prior studies [13, 20], these data suggest a selective vulnerability of endothelial cell miRNAs to AD pathology.

miRNAs and their targets indicating biological sex differences

Almost half (27; 44%) of mapped miRNAs indicated statistically significant differences among males and females prior to AD. Further, most (22; 82%) of those miRNAs were significantly higher in expression in females relative to males. With considering that miRNAs are negative regulators of mRNA expression and transcription, this would yield a general downregulation of respective mRNA targets in females more so than males. A key subset of differential markers among males and females as shown in Table 4 include voltage-gated Na⁺ channels (e.g., SCN2B), synaptotagmins (e.g.,SYT1), and histone deacetylases (HDACs; e.g., HDAC8, SIRT1). SCN2B impairment and/or deletion underlies atrial and ventricular arrhythmias [61], whereas aberrant SCN2A and SCN8A expression/function is related to epilepsy and/or autism spectrum disorder [62]. SYT1 and SYT2 mediate Ca²⁺-dependent neurotransmitter release and have been identified as therapeutic targets for syndromic intellectual disability [63] and myasthenia gravis [64] respectively. Thus, it is possible that SYT may regulate different functions in males and females, depending on tissue and cell type, and the presence or absence of sex hormones in accord with animal age and/or pathology. HDACs and their role in protein acetylation have been implicated in an array of neurodegenerative disease pathologies as well such as Parkinson’s disease, Huntington’s disease, and AD [65]. With regard to females, HDAC8 and SIRT1 in particular are co-regulators of nuclear estrogen-related receptor α expression and its DNA-binding affinity [66]. As a key characteristic of breast cancer, heightened estrogen signaling can increase HDAC8 expression [67], which contributes to aggressiveness of tumor growth and spread in tandem with resistance to chemotherapy [68]. Another interesting observation entails how KCNMB1 [codes for β subunit of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels] was also a strong indicator among males and females. It has been recently demonstrated that depletion of estrogen indeed impairs vasodilatory activity of cerebral arterioles and functional hyperemia of the brain via reduced BK_Ca channel function [69]. Overall, the precise mechanisms for how biological sex plays a role in setting the stage for early AD pathology remains a ripe topic for future investigation.

Study considerations and future directions

The current study represents an in silico follow-up analysis to an original study [10] that did not examine age and sex-matched B6129SF2/J mice as the background wild-type strain for the 3xTg-AD animals of study. However, we reinforce that the significance of our miRNA data pertains to early development of AD up to 8 months at the oldest age point, whereby cerebrovascular structure [70], neurovascular function [71], and cognition [27, 72, 73] remain relatively stable in wild-type animals. Further, with n-values of 3 per individual study group for males and females, future studies will be required for completely resolving roles of biological sex and/or subtle pathological phases throughout the healthspan/lifespan of the 3xTgAD mouse. Further, note that behavioral features (well-known, characterized) may not necessarily be reflective of comprehensive changes in molecular expression (mostly unknown or unclear) with absolute precision over time. With molecular events likely being the driver of disease, the original histological and behavioral evidence provided a starting point for examining gene profiles during the life of the 3xTgAD mouse.

Based on our current data set for biological sex for example, we will refine focus on early stage disease only in future experiments with transition to cognitive impairment and initial deposits of extracellular amyloid in the brain. As one example, we are interested in how testosterone can potentially upregulate let-7d [74] during early development of AD. With notable roles for markers such as HDAC8 and VEGFA, another future objective is to quantify a potential overlap of preclinical dementia and cancer pathology at the molecular level. All discussed mRNA markers and respective pathways for indicating development of cerebrovascular aging and AD will require thorough experimental confirmation using molecular biology, ex vivo, and in vivo study tools in future studies.

SUMMARY AND CONCLUSIONS

Optimal neurovascular function is required for healthy cognition throughout life, whereby its disruption can set the stage for relatively widespread types of dementia such as AD. The molecular and integrative underpinnings of AD pathology remain poorly understood overall while extremely complex and thereby requiring comprehensive methods of analysis. Based on our original data collected for cerebrovascular miRNA expression throughout the healthspan of 3xTg-AD mice, we conducted an indepth in silico analysis of relevant mRNA markers along cardiovascular and nervous system signaling pathway strings. A total of 61 miRNAs were mapped, whereby most (≈70%) of cardiovascular markers are integrated with the nervous system throughout miRNAs. Common targets among miRNAs that are significantly altered in expression during AD conditions entail those that govern angiogenesis (e.g., PDE, PDGF, and SMAD families) while underscoring molecular markers of cellular excitotoxicity (e.g., GRIN2B, SLC6A1, SYT3) and inflammation (e.g., TNF family). Most differences noted among males and females are prior to AD and center on markers that modulate membrane excitability for cardiac contractility and neurotransmission (e.g., SCN and SYT families), transcriptional regulation and protection of the proteome (e.g., HDAC8, HSPA1A/1B, SIRT1), and cell cycling and proliferation (e.g., ERBB3, UBE2G1/V1). Altogether, these data provide a comprehensive molecular “fingerprint” of the early progression of AD pathology based on neurovascular pathway analyses of cerebral blood vessels in the brain of an animal study model. In such manner, this study may provide valuable information that will successfully inspire and direct future translational and clinical studies of AD using novel vascular miRNA and mRNA targets for diagnosis and therapy.

DATA AVAILABILITY

The data that support the findings of this study are available from the corresponding author upon reasonable request.